Minyoung Park February 2011 - Stackstc750bj0003/Feb... · 2011-09-22 · and Theodore S. Jardetzky...

DEVELOPING A PROTEIN-BASED ASSAY FOR IDENTIFYING

HRSV ENTRY INHIBITORS AND KNOWLEDGE-BASED

APPROACHES TO DESIGN PEPTIDOMIMETICS

A DISSERTATION

SUBMITTED TO THE DEPARTMENT

OF CHEMICAL AND SYSTEMS BIOLOGY

AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD

UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQURIREMENTS

FOR THE DEGRESS OF

DOCTOR OF PHILOSOPHY

Minyoung Park

February 2011

http://creativecommons.org/licenses/by-nc/3.0/us/

This dissertation is online at: http://purl.stanford.edu/tc750bj0003

© 2011 by Min Young Park. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.

ii



http://purl.stanford.edu/tc750bj0003

I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.

Annelise Barron, Primary Adviser


Theodore Jardetzky, Co-Adviser


James Chen


Jennifer Cochran


Thomas Wandless

Approved for the Stanford University Committee on Graduate Studies.

Patricia J. Gumport, Vice Provost Graduate Education

This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.

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v

Abstract

Biotherapeutics have regained their reputation as drug candidates in the drug

market due to their remarkable specificity; however, the broader clinical use of

biotherapeutics is often challenged by poor pharmacological properties. Therefore,

there is enormous interest in developing peptidomimetics as alternative therapeutic

options.

Inspired by the first successful example of peptide-based antivirals, Fuzeon®,

similar strategies to develop antivirals have been applied to many other viruses that

share class I fusion protein-mediated viral fusion. Using human Respiratory Syncytial

Virus (hRSV) as a model system, the second chapter of this dissertation demonstrates

our successful effort to develop a protein-based assay using a 5-Helix Bundle (5HB)

fluorescence polarization (FP) as a screening platform for hRSV fusion inhibitors. The

remaining chapters in this thesis all utilize the 5HB-based FP assay to evaluate the

potential of short peptides and their peptoid-based peptidomimetics as antivirals to

control hRSV infections.

The third chapter proposes that NMEGylation, an alternative to PEGylation

that uses a covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain,

may enhance the bioavailability of short therapeutic peptides. The incorporation of

optimized numbers of NMEG monomers along with a glycine linker increases the

solubility and serum stability greatly, suggesting that NMEGylation may open a new

opportunity to use peptoids as modifiers of therapeutically attractive peptides and

proteins.

vi

The fourth chapter demonstrates how our novel approach combining alanine,

proline, and sarcosine scans can be useful to determine peptoid-replaceable peptide

residues and further proves the usefulness of the combined scan strategy using the C20

peptide as a parent peptide. Furthermore, two different methods to promote the !-

helical conformation of C20 analogues by structurally constraining the parent C20

peptide using “hydrocarbon-stapling” or “clicking” are described. We report a

constrained C20 analogue with improved binding affinity, and discuss our structural

investigations of the constrained C20 analogues using CD spectroscopy.

The studies in the fifth chapter show that phage display can be used for

identifying novel hRSV entry inhibitors that are not derived from the original

sequence of the hRSV fusion protein F like the peptides described in chapters 2, 3, and

4. We report two 12-mer peptides with a low micromolar binding affinity to the 5HB,

and ongoing efforts are aimed towards better understanding the interaction of the 12-

mer peptides with the 5HB by co-crystallizing the peptides and 5HB.

Finally, the dissertation concludes with a sixth chapter that summarizes the

current status of each research chapter. In addition, future prospects, new directions,

and potential applications of the findings in this dissertation are presented in this

chapter.

vii

Acknowledgments

Graduate school has been a long but indeed incredible journey to me. I feel so

blessed to have such great friends and colleagues who have helped me so much to get

through many ups and downs that I have faced during all those years in graduate

school. I have learned how to be a good scientist and colleague, and most importantly,

a human being from them. Without these helps, I know I could not be here to describe

how thankful I am.

I am grateful to acknowledge that my advisors, Professors Annelise E. Barron

and Theodore S. Jardetzky who have pushed me to become a better and more

independent scientist. I will always keep in mind invaluable training and experience

they allowed me to have throughout these years. I also want to show my deep

gratitude to the committee members: Professors Tom Wandless, James Chen and

Jennifer Cochran, who greatly supported and guided my research project.

I feel deeply indebted to Dr. Modi Wetzler as my scientific mentor as well as a

wonderful friend. On the top of kindness and smartness, he is an inspiring and very

thoughtful scientist. Drs. Jiwon Seo and James Broering from the Barron group and

Dr. Beth Wurzberg from the Jardetzky group also have been great colleagues and

friends. With their help, countless mistakes that I have made could be easily converted

into valuable knowledge without too much pain. I have learned so much from them,

and can only hope to be a mentor like them to someone sooner or later.

I would like to thank my former and current lab-mates from both the Barron and

Jardetzky labs who have become my friends and family. With their friendship and

support, my life in graduate school could be enjoyable after all.

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The most importantly, I am deeply thankful to my mom, dad, and sister for their

constant and unconditional love and support. I could come this far because I always

knew that my family would be standing by me no matter what happens.

And lastly, YOU! You are my inspiration!

ix

Table of Contents

Abstract """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" v

Acknowledgements """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" vii

List of Tables """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" xiii

List of Figures """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" xv

List of Schemes """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" xx

List of Equations """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" xx

Chapter One: Introduction

Synthetic peptide therapeutics: Inspiration from Fuzeon """""""""""""""""""""""""""""""" 1

Class I viral fusion proteins """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 2

Class I viral fusion protein-mediated membrane merger """""""""""""""""""""""""""""""" 4

Shortcomings of peptide therapeutics: A lesson from Fuzeon """""""""""""""""""""""" 5

Recombinant proteins to overcome challenges in biotherapeutics """"""""""""""""" 6

General approaches to design peptidomimetics """""""""""""""""""""""""""""""""""""""""""""" 7

Peptidomimetic folding oligomers: Foldamers """"""""""""""""""""""""""""""""""""""""""""""" 9

Peptoid biomimicry """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 10

Challenges in transforming therapeutic peptides to peptoids """"""""""""""""""""""""" 12

Overview of the thesis """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 14

References """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 17

Chapter Two: Design and evaluation of a structure-guided screening

platform for peptide-based hRSV entry inhibitors

Human Respirator Syncytial Virus (hRSV) """"""""""""""""""""""""""""""""""""""""""""""""""" 26

Urgent need to develop hRSV treatment """""""""""""""""""""""""""""""""""""""""""""""""""""""" 28

x

Current hRSV treatment options """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 29

Current options for hRSV prevention """""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 30

Development of screening methods for hRSV fusion inhibitors """"""""""""""""""" 31

5-Helix Bundle (5HB) construct design, expression, purification

and secondary structure analysis """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 33

Validation of 5HB as an hRSV F protein 6HB mimic by ELISA assays """""" 35

Saturation binding FP measurement """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 36

Specificity of Fl-C35 binding to the 5HB """"""""""""""""""""""""""""""""""""""""""""""""""""""" 38

Competitive FP assays """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 39

Suitability as a high-throughput screening system """""""""""""""""""""""""""""""""""""""" 41

Small molecule fusion inhibitors tested in the 5HB-based "

competitive FP assays """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 43

Conclusions and future prospects """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 45

Materials and methods """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 47

References """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 53

Chapter Three: NMEGylation, a novel modification to enhance the

bioavailability

Limitations in using synthetic peptides as therapeutics """""""""""""""""""""""""""""""""" 60

PEGylation and its pressing challenges """""""""""""""""""""""""""""""""""""""""""""""""""""""""" 60

A novel peptide/protein modification: NMEGylation """""""""""""""""""""""""""""""""""" 62

Synthesis, purification, and characterization of NMEGylated

C20 peptides """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 65

Biological activity of NMEGylated C20 peptides """"""""""""""""""""""""""""""""""""""""""" 67

Serum stability of NMEGylated C20 peptides """""""""""""""""""""""""""""""""""""""""""""""" 69

Secondary structure analysis of NMEGylated C20 peptides

in different solvent systems """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 70

Conclusions and future prospects """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 74

xi

Materials and methods """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 75

References """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 81

Chapter Four: Knowledge-based approaches for designing peptoid-

peptide hybrids and their structurally constrained analogues

Background and motivation for this study """""""""""""""""""""""""""""""""""""""""""""""""""""" 87

A combined approach of alanine, proline, and sarcosine scan """"""""""""""""""""""" 88

Alanine substitution study """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 91

Proline substitution study """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 93

Sarcosine substitution study """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 95

Synthesis and characterization of peptomeric C20 analogue """"""""""""""""""""""""""" 97

Importance of !-helices in biological settings """""""""""""""""""""""""""""""""""""""""""""" 100

Structurally constrained C20 peptide derivatives via hydrocarbon

stapling """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 101

Structurally constrained peptomers using click chemistry """""""""""""""""""""""""""" 105

Conclusions and future prospects """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 111

Materials and methods """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 114

References """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 122

Chapter Five: Identifying short peptide-based hRSV entry inhibitors from

a phage-displayed peptide library and their peptidomimetic analogues

Overview of bacteriophages """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 129

Phage display technology """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 130

Applications in phage display technology """""""""""""""""""""""""""""""""""""""""""""""""""" 133

hRSV F protein as a target for phage-displayed peptide library """"""""""""""""""" 135

Phage-displayed peptide library panning results and ELISA tests """"""""""""""" 136

Competitive FP assays of selected synthetic peptides """""""""""""""""""""""""""""""""" 138

Secondary structure analysis of selected synthetic peptides """"""""""""""""""""""""" 141

xii

Efforts to cocrystallize selected peptides with the 5HB """"""""""""""""""""""""""""""" 142

Alternative ways to cocrystallize selected peptides with the

GCN constructs """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 144

Preliminary data for antiviral activities of selected peptides """""""""""""""""""""""" 146

Peptomeric hRSV inhibitors and their binding activity toward the 5HB """"" 147


Materials and methods """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 151

References """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 157

Chapter Six: Conclusion


References """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 171

xiii

List of Tables

Chapter Two

Table 2-1. Peptides derived from the HRB domain of hRSV F protein

and their sequences and binding affinities to the 5HB """"""""""""""""""""""" 40

Table 2-2. Human respiratory syncytial virus fusion inhibitors """""""""""""""""""""""""" 43

Chapter Three

Table 3-1. Marketed PEGylated biotherapeutics """""""""""""""""""""""""""""""""""""""""""""""""" 61

Table 3-2. NMEGylated C20 peptomer sequences tested in this study

with molecular weight (MW), purity, solubility, hydrophilicity

and their IC50 values """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 66

Chapter Four

Table 4-1. Sequences of alanine-substituted peptides tested in this study

and their inhibitory effect on binding of Fl-C35 to the 5HB """""""""""""""" 91

Table 4-2. Sequences of proline-substituted peptides tested in this study


Table 4-3. Sequences of sarcosine-substituted peptides tested in this study


Table 4-4. Ratio of molar ellipticities (deg·cm2·dmole

-1) at 222 and 208, and

percent helicity calculated for stapled peptides and clicked

peptomers """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""103

xiv


-1) at 222 and 208, and

percent helicity calculated for stapled peptides and clicked

peptomers """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""109

Chapter Five

Table 5-1. Sequences of phage-bound peptides isolated by screening

against the 5HB """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""136

Table 5-2. Peptides individually synthesized and used in this study """"""""""""""""""139

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List of Figures

Chapter One

Figure 1-1. Schematic diagram of class I viral fusion proteins """""""""""""""""""""""""""""" 2

Figure 1-2. 6-helix bundle structures of class I viral fusion proteins """"""""""""""""""""" 3

Figure 1-3. Proposed model of class I viral fusion protein-mediated

virus entry """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 4

Figure 1-4. Multimerized helical bundles to mimic 6HB of

class I viral fusion proteins """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 7

Figure 1-5. Sequence-specific peptidomimetic oligomers """"""""""""""""""""""""""""""""""""" 9

Figure 1-6. Structure comparison between peptoids and peptides """""""""""""""""""""""" 10

Figure 1-7. Submonomer approach for peptoid synthesis """"""""""""""""""""""""""""""""""""" 11

Figure 1-8. Secondary structures of Peptoids """""""""""""""""""""""""""""""""""""""""""""""""""""""" 11

Figure 1-9. Example of mimicking protein functions using

peptoid helical bundles """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 12

Figure 1-10. Rapid trans- and cis-isomerization in peptoid backbone """"""""""""""""""" 13

Chapter Two

Figure 2-1. Schematic diagram of hRSV fusion (F) protein """"""""""""""""""""""""""""""""" 26

Figure 2-2. X-ray crystal structure of hRSV 6HB """""""""""""""""""""""""""""""""""""""""""""""" 27

Figure 2-3. A key interaction between HRA and HRB helices

and the hydrophobic pocket formed by neighboring

HRA helices in the 6HB assembly """"""""""""""""""""""""""""""""""""""""""""""""""""" 30

xvi

Figure 2-4. An illustration of hRSV 5-Helix Bundle (5HB)

with a resulting SDS-PAGE of 5HB purification """""""""""""""""""""""""""""" 33

Figure 2-5. Secondary structure analysis and thermal stability

of 5HB by CD spectroscopy """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 34

Figure 2-6. A schematic diagram of Trx-C49 with a resulting

SDS-PAGE of purified Trx-C49 """"""""""""""""""""""""""""""""""""""""""""""""""""""""" 35

Figure 2-7. Indirect and competitive ELISA assays """"""""""""""""""""""""""""""""""""""""""""" 36

Figure 2-8. Binding titration curve of Fl-C35 and its binding stability

to the 5HB """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 37

Figure 2-9. Specificity and selectivity of Fl-C35 to the 5HB """""""""""""""""""""""""""""""" 39

Figure 2-10. Competitive FP assays of unlabeled C35 and N- and C-terminally

truncated peptides """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 41

Figure 2-11. Assay robustness test by FP measurements of free and

bound Fl-C35 controls """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 42

Figure 2-12. Structures of hRSV fusion inhibitors tested in this study """""""""""""""""" 43

Figure 2-13. Competitive FP assays of small molecule fusion inhibitors """""""""""""" 44

Chapter Three

Figure 3-1. Structure of N-methoxyethylglycine (NMEG) and

poly-ethylene glycol (PEG) """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 62

Figure 3-2. Synthetic approaches to prepare peptoids, peptides and hybrids """""" 64

Figure 3-3. Resulting data from competitive FP assays """""""""""""""""""""""""""""""""""""" 67

xvii

Figure 3-4. Optimization of the length of the glycine linker """""""""""""""""""""""""""""""" 68

Figure 3-5. Peptide stability in presence of serum """""""""""""""""""""""""""""""""""""""""""""""" 70

Figure 3-6. Comparison of helical propensity of NMEGylated

C20 analogues with C20 peptide at 222 nm """""""""""""""""""""""""""""""""""""""""" 71

Figure 3-7. Solvent polarity dependency in CD spectra

of 100 µM of C20NMEG3 """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 72

Figure 3-8. CD spectra of different concentrations of C20NMEG3

ranging from 10 to 200 µM in various solvent systems """""""""""""""""""""" 73

Chapter Four

Figure 4-1. Structural comparison of alanine, proline, and sarcosine """"""""""""""""""" 89

Figure 4-2. % Inhibition of alanine-substituted peptides at 100 and 200 µM """"""" 92

Figure 4-3. % Inhibition of proline-substituted peptides at 100 and 200 µM """"""" 94

Figure 4-4. % Inhibition of sarcosine-substituted peptides at 100 and 200 µM """" 97

Figure 4-5. (A) Structures of peptoid residues corresponding to Glu

and Lys residues, respectively and (B) structure of NLysNGluC20

peptomer """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 98

Figure 4-6. Competitive FP assay of NLysNGluC20 """"""""""""""""""""""""""""""""""""""""""""" 99

Figure 4-7. Predicted stabilized helical conformations by hydrocarbon stapling

(A) SUMP1 and (B) SUMP2 """""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 101

Figure 4-8. CD spectra of (A) SUMP1 and (B) SUMP2 """"""""""""""""""""""""""""""""""""" 102

Figure 4-9. Competitive FP assay of SUMP2 """""""""""""""""""""""""""""""""""""""""""""""""""""" 104

xviii

Figure 4-10. Predicted stabilized helical conformation of the C20 peptide

by click reaction """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 105

Figure 4-11. Structures of peptoid residues with clickable functional groups """""" 106

Figure 4-12. Structures of (A) C20_C_U and (B) C20_C_C with desired

molecular weights """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 107

Figure 4-13. CD spectra of (A) C20_C_U and (B) C20_C_C """"""""""""""""""""""""""""""""" 108

Figure 4-14. Competitive FP assay of C20_C_U and C20_C_C """"""""""""""""""""""""""""" 109

Figure 4-15. Helical wheel representation of the C20 peptide """""""""""""""""""""""""""""""" 111

Figure 4-16. Comparison of %Inhibition and helical propensity """""""""""""""""""""""""" 112

Chapter Five

Figure 5-1. Schematic diagram of a filamentous phage (M13) """"""""""""""""""""""""""" 129

Figure 5-2. Summary for obtaining high-affinity peptide ligand

for binding to target molecules using phage-displayed

peptide libraries """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 132

Figure 5-3. Concept of mirror-image phage display """""""""""""""""""""""""""""""""""""""""""" 134

Figure 5-4. Binding of selected phage-bound peptides to immobilized 5HB """"" 137

Figure 5-5. % Inhibition of Fl-C35 binding to the 5HB by resynthesized

peptides using a competitive FP assay """""""""""""""""""""""""""""""""""""""""""""" 140

Figure 5-6. Competitive FP assays of P1-5 and P1-8 """""""""""""""""""""""""""""""""""""""""" 140

Figure 5-7. CD spectra of (A) reference spectra of secondary structures and

(B) P1-5 and P1-8 """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 142

xix

Figure 5-8. Two different handedness of the 5HB bundling """"""""""""""""""""""""""""""" 143

Figure 5-9. (A) Schematic diagram of GCN constructs (HG and GHG)

for crystallization study and (B) illustration of trimerized

GCN constructs """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 144

Figure 5-10. FP response of GCN constructs in the presence of Fl-C35

peptide """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 145

Figure 5-11. Peptides (P1-5 and P1-8) inhibition of hRSV infection

in Hep2 cells """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 147

Figure 5-12. Structures of peptoid residues to replace proline in P1-8 """"""""""""""""" 148

Figure 5-13. Structures of P1-8 mimics """"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 149

Figure 5-14. Resulting data of P1-8 mimics using a competitive FP assay """"""""""" 149

Chapter Six

Figure 6-1. Stabilized the binding of NmegGC20 to the 5HB by H-bond """"""""""" 166

Figure 6-2. Retaining biological activity of NMEGylated ligands

and steric hindrance """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 167

Figure 6.3 Schematic diagram of (A) a scaffold for trimeric HIV-1 gp41

mimetics and (B) the resulting trimeric helix bundle """"""""""""""""""""""" 169

Figure 6.4 Structures of (A) Tris-(bromomethyl) benzene (TBB) and

(B) Tris-(2-aminoethyl)amine (TREN) """"""""""""""""""""""""""""""""""""""""""""" 169

Figure 6.5 Schematic representations of peptide dendrimer motif examples """"" 170

xx

List of Schemes

Chapter Two

Scheme 2-1. Strategy designed and developed in this study """"""""""""""""""""""""""""""""""" 32

Chapter Four

Scheme 1. Synthesis of 3-azidopropylamine """""""""""""""""""""""""""""""""""""""""""""""""""""" 116

Scheme 2. Microwave-assisted Cu (II) catalyzed click chemistry on resin """"""" 118

Scheme 3. Modified Kaiser method to determine the completion of the click

reaction """""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""" 120

List of Equations

Chapter Two

Equation 2-1. Equation for calculating Z’ factor """""""""""""""""""""""""""""""""""""""""""""""""""""" 42

1

Chapter One

Introduction

Synthetic peptide therapeutics: Inspiration from Fuzeon

Even with the knowledge gained after tens of billions of dollars spent on small

molecule drug discovery, there is a rather disappointing success rate of 3% for

reaching preclinical development and 7% for successful launching in the market from

clinical trials.1,2 Consequently, synthetic peptides have regained their standing as

potential drug candidates and the biotherapeutic market has been rapidly growing.

Clinically-available synthetic peptides have proven their undeniable merits as

therapeutics including strong binding affinity, high specificity and selectivity,

minimized and often predictable drug-drug interaction profiles and lower toxicity.3,4

Because of these attractive attributes of peptides as drug candidates, there are

approximately 700 peptides in the development stage worldwide including around 270

peptides in clinical phases and about 400 in advanced preclinical phases.3,5

Additionally, more than 60 synthetic peptides have already reached the market with

sales reaching US $40 billion.3,4

Among the FDA-approved peptide-based drugs, Fuzeon® (36-amino acid-

long, enfuvirtide, Trimeris/Roche), a widely known anti-HIV agent, has been

particularly inspiring to us because it represents the first case of a peptide medicine

derived from a viral protein, HIV-1 gp41.6-8 Due to its low nanomolar antiviral activity

and the urgent need to develop HIV treatments, Fuzeon® (T-20 before FDA approval)

Chapter One: Introduction 2

was granted fast track status for FDA-approval and became the first viral entry

inhibitor targeting HIV-1 gp41-mediated viral fusion.8-10 Since the product launch in

2003, worldwide sales of Fuzeon® have reached US $170 million in 2008, even with

a slight sales drop compared to 2007.11 The comparatively low sales of Fuzeon ®

(“blockbuster” drugs typically have annual sales of > US $1 billion) reflects its use as

a drug of last resort for resistant HIV-1 infections due to its high cost and injection

delivery method. In any case, the great success of Fuzeon® as a new class of antiviral

agents has made class I viral fusion proteins, which HIV-1 gp41 belongs to, attractive

antiviral targets.

Class I viral fusion proteins

Class I viral fusion proteins are frequently found in many virus families

including paramixoviridae, e.g., parainfluenza virus 5 (PIV5) and human respiratory

syncytial virus (hRSV); retroviridae, e.g., HIV-1; coronaviridae, e.g., severe acute

respiratory syndrome coronavirus (SARS-coV), and orthomyxociridae, e.g., influenza

(A) Paramyxovirus F

(B) HIV-1 gp41

Figure 1-1. Schematic diagram of class I viral fusion proteins (A) The paramyxovirus fusion protein (F) and (B) HIV glycoprotein (gp) are shown as representatives of class I viral fusion proteins. Fusion peptide (FP) and transmembrane domains (TM) are located adjacent to two heptad repeat regions (HRA and HRB). Images are adapted from Lamb et

al. (2007) Curr. Opin. Struc. Biol. 17, 427


virus12 and share similar structural and functional features. As shown in Fig. 1-1, these

fusion proteins are first synthesized as intact precursor proteins and then cleaved by

furin-like proteases, yielding two subunits and subsequently forming a trimeric

conformation. Two heptad repeat regions (HRA and HRB) are located adjacent to a

fusion peptide (FP) and a transmembrane domain (TM) and contain a strong coiled-

coil motif. Class I viral fusion proteins are believed to be a primary driving force for

the membrane merger between virus and host cell through the formation of an

extremely stable 6-helix bundle (6HB). 3-D structures of these proteins determined by

X-ray crystallography reveal remarkable structural similarity to each other, where the

three HRA helices assemble into an inner core, while the three HRB helices pack

against this trimeric inner core in an anti-parallel manner, eventually forming the 6HB

(Fig.1-2).

(A) hRSV F

(B) HIV-1 gp41

Figure 1-2. 6-helix bundle structures of class I viral fusion proteins Core structures of (A) hRSV fusion protein F and (B) HIV-1 gp41 are illustrated from the corresponding X-ray crystal structures, showing striking similarities.


Class I viral fusion protein-mediated membrane merger

Viral entry is the most essential step during the infectious cycle, and is an

attractive drug target because it occurs extracellularly, providing relatively easy access

to antivirals. Based on pre- and post-fusion structures of PIV5 and HPIV3,

respectively, determined by the Jardetzky laboratory, a mechanism of paramyxovirus

fusion protein F-mediated membrane merger has been proposed (Fig. 1-3).13 Upon

activation by viral attachment, the F protein assembles into a pre-fusion form, then an

intermediate conformation and finally undergoes a dramatic conformational change to

a highly stable 6HB as shown in Fig. 1-3.13-15 Since Fuzeon® has proved the

therapeutic value in blocking this 6HB formation to prevent viral fusion and further

viral infection, disruption of the 6HB assembly by peptides derived from viral fusion

proteins has been a successful strategy for antiviral drug discovery, yielding several

promising viral entry inhibitors currently in clinical trials against viruses that share

class I viral fusion proteins such as hRSV and SARS-coV.16-19

Virus

Host cell Figure 1-3. Proposed model of class I viral fusion protein-mediated virus entry, based on structural studies by X-ray crystallography (Image adapted from Yin et al.

(2006) Nature 439, 38) HRA and HRB domains are in green and blue, respectively. Intermediate stage is believed to last several minutes, thereby becoming an attractive antiviral target. Fuzeon® and other peptide antivirals target this stage to prevent 6HB formation and further viral entry.


Shortcomings of peptide therapeutics: A lesson from Fuzeon

Challenges in using synthetic peptides in clinics still remain because of high

manufacturing cost and poor biophysical properties, including rapid renal clearance,

low solubility, and limited thermal stability.20-22 In general, peptides require a certain

length to form proper secondary structures such as !–helix or "-turn in aqueous

solution and once peptides adapt these distinctive structures, they become functional

and less sensitive to proteolytic degradation. Short and therefore unstructured peptides

(15–25 aa in length) are highly vulnerable to proteases, resulting in short half-lives (2–

5 min).23,24 On the other hand, longer peptides can be more structured with relatively

low susceptibility to proteases, but more complicated synthesis steps are required for

longer peptides, which greatly increase manufacturing costs. In addition, the variable

solubility, limited stability, low cell-permeability, and high immunogenecity of

peptides still restrict their development and the usage in clinic.

For example, Fuzeon®, a 36 amino acid-long peptide, requires 106 chemical

synthesis steps, resulting in a high cost of production and treatment

(~$30,000/year/patient in the United States).25,26 Additionally, due to low

bioavailability, Fuzeon® cannot be orally delivered and needs to be administered

twice daily via injection with high dose (90 mg/day), leading to potential side effects

as well as poor patient compliance.6,27 Even with such a high efficacy as an anti-HIV

agent, Fuzeon®’s high cost and complex administration have restricted its overall

growth worldwide as previously discussed.


Recombinant proteins to overcome challenges in biotherapeutics

To circumvent the limitations of synthetic peptides, most of the top 100 FDA-

approved biotherapeutics are large or highly structured, accounting for their enhanced

chemical, physical and thermal stability.6 Similarly, instead of using small helical

protein segments derived from class I viral fusion proteins, larger recombinant

proteins containing multiple protein fragments have been designed and investigated as

viral fusion inhibitors. As the first example of this approach, the 5-Helix was created

by alternately linking three HRA helices and two HRB helices from the HIV-1 gp41

with short peptide linkers (Fig. 1-4A). This recombinantly engineered protein

construct shows extreme thermal stability with high !-helical content.28 The 5-Helix

was initially created as an HIV entry inhibitor; however, it was also tested as a target

for screening small molecule anti-HIV-1 agents.28,29

In the case of hRSV, multimerized HRA and HRB helices (HR121 and

HR212) were designed from hRSV F protein and tested as anti-hRSV agents (Fig. 1-

4B), showing nanomolar activity in cell-cell fusion assay.30 This exact strategy has

also been applied to HIV-1 gp4131 by the same research group, highlighting that these

multimerized protein inhibitors have great potential to be successful antiviral agents

with high antiviral activity. Although these recombinant proteins seem to overcome

peptides’ intrinsically poor biophysical properties, there are still unavoidable problems

that need to be solved. The molecular sizes of proteins are generally orders of

magnitude larger than traditional small molecule drugs, complicating drug delivery

and necessitating delicate storage condition with poor shelf-life that make proteins less

attractive as pharmaceuticals.32


General approaches to design peptidomimetics

Recent revolutionary advances in biotechnology have yielded significant

success in prolonging the half-lives of synthetic peptides, often utilizing a new class of

therapeutically attractive molecules called peptidomimetics. Peptidomimetics can

imitate the desired elements of peptides and alter undesirable properties such as

proteolytic susceptibility, while more importantly retaining the biological functionality

A

B

Figure 1-4. Multimerized helical bundles to mimic 6HB of class I viral fusion proteins (A) 5-Helix derived from HIV-1 gp4 and (B) HR121 and HR212 derived from HIV-gp41 and hRSV F proteins


of the peptides of interest.33 The traditional strategy to design therapeutic

peptidomimetics starts with biologically active peptides that bind to the target

molecules. Once either biologically-derived or synthetically-generated peptides of

interest are chosen, these peptides can serve as a template to design

peptidomimetics.34-36

Typically, after critical residues for biological functions of peptides are

identified by the conventional methods such as truncation and deletion studies and an

alanine scan,3,6,21,22,37,38 the resulting optimized peptide sequences can be further

refined with the replacement of each amino acid by D-amino acids or unnatural amino

acids to define conformation parameters (e.g., chirality, !-helicity, and H-bonds).

These parameters allow us to better understand crucial conformations of the biological

active core in target peptides, presumably providing the possibility of the modification

in the peptide structure to improve the peptides’ pharmacokinetic properties, reduce

the degree of proteolytic degradation, and thereby extend the half-lives.33

One representative example of such peptide modifications is structurally

rigidifying peptides by creating chemical linkages between residues (e.g., stapled

peptides,39,40 bicyclic peptides,41,42 and chemical crosslinking43). Since short peptides

are generally unstructured, the entropy loss upon binding to target proteins reduces

affinity. To minimize this entropy penalty, efforts to introduce conformational

constraints to peptides have resulted in promising therapeutic peptidomimetics with

substantially enhanced biological activity for the desired target.33,36 These

peptidomimetics are highly protease-resistant, and have reduced immunogenecity as

well as improved bioavailability compared to the parent peptides. As the market for


peptide-based drugs has greatly expanded, peptidomimetics research has also grown

using non-peptidic chemicals,44,45 or peptidic molecules to mimic structural feature of

peptides while biological activities are retained or even enhanced.3,5,22

Peptidomimetic folding oligomers: Foldamers

In addition to peptide mimics that greatly alter the backbone structure such as

azapeptides46 and oligoureas (Fig. 1-5),47 peptidomimetic folding oligomers, known as

foldamers, have received considerable attention because this class of peptidomimetics

exhibits more versatile secondary structural elements including both !-helices and "-

turns. Foldamers are composed of simple unnatural building blocks as shown in dotted

NH

NHNN

R1O

O

NH

R2

OHN

HN

NH

NH

O

R1 O

NH

NH

O O

NH

R1 R2

NH

R1

NH

O O

NH

R2

NH

R1 HN

O

NH

R2

O

NN

N

O

O

R1

R2

R3

D. !3-PeptideC. !2-Peptide

E. "-Peptide F. Peptoid

A. Azapeptide B. Oligourea

Figure 1-5. Sequence-specific peptidomimetic oligomers (A repeating unit marked in dotted boxes)


(A) (B)

HN

OH

OR

HN

OH

OH

Rn n

Figure 1-6. Structure comparison between (A) peptoids and (B) peptides

boxes in Fig. 1-5, which greatly increase biostability while effectively mimicking

protein conformations and interactions.48,49 Moreover, foldamers can be efficiently

synthesized with chemically diverse monomers in a cost-effective manner and can be

designed to be more water soluble, biocompatible and conformationally even more

stable, depending on the specific monomers incorporated.50 Among the many types of

foldamers, helical foldamers including "-peptides,51-53 #-peptides,54,55 and N-

substituted glycines (peptoids)56-58 are relatively well characterized and their

applications as therapeutic candidates are being actively investigated (Fig. 1-5).

Peptoid biomimicry

Peptoids are non-natural, but

easily synthesized and sequence-

specific oligomers of N-substituted

glycine. Peptoids are structural isomers

of peptides where the side chains are

attached at nitrogen atoms instead of carbon atoms (Fig. 1-5F and 1-6). This

isomerism leads to several important consequences: there are no hydrogen bond

donors in peptoids, and the backbone is achiral and more flexible. However, by

incorporating selected !-chiral bulky side chains, we can create the conformation of

the polyproline type I-like (PPI) helical structure,59,60 with controlled handedness. An

efficient “submonomer” approach using primary amines as side chains with an

automated solid-phase synthesis (Fig. 1-7)61,62 has spurred a number of research

groups to use peptoids in their research. These efforts have expanded the scope of


A B C

Figure 1-8. Secondary structures of Peptoids NMR structure of (A) a peptoid threaded

loop, (B) a "-hairpin mimicking peptoid, and (C) X-ray crystal structure of a cyclic peptoid,

highlighting the peptoid backbone in green.

NH2

Br

O

DIC/DMFNH

Br

O

HO

NH

O

NH

H2N R1

R1

N

O

NH

R1

NH

O R2n

Repeat to desired length

NMP

Figure 1-7. Submonomer approach for peptoid synthesis

peptoid research from the fundamental understanding of the intermolecular

interactions in a simple helical mimicking structure to the potential therapeutic

applications of peptoids in a highly structured molecular environment.35,50,58,60

To better understand peptoids as biomimetic material, the relationship between

peptoid secondary structures and side chains,57,63 and structural analyses on linear

(e.g., thread-loop64, "-hairpin-like peptoid,65 and PPI-helix59,66-68) and cyclic

peptoids69,70 have been extensively investigated using CD, NMR and X-ray

crystallography, yielding valuable profound knowledge on peptoids (Fig. 1-8).


Furthermore, the importance of tertiary structures in highly structured proteins

should be considered, because specific functions of individual proteins are mostly

determined by their tertiary structures. Therefore, the tertiary structure of peptoids has

also been investigated by studying the bundling of amphiphilic peptoids71 and the self-

assembly of peptoid-based helical bundles for mimicking protein helical bundles

including Zn-binding metalloproteins (Fig. 1-9).72,73

Challenges in transforming therapeutic peptides to peptoids

Even with these recent accomplishments in peptoid research, there are very

few successful studies on developing peptoids as binding partners (e.g., antagonist or

agonist) targeting therapeutically important protein-protein interactions. A simple way

to convert biologically active peptides to peptoid analogues can be the substitution of

Figure 1-9. Example of mimicking protein functions using peptoid helical bundles Two peptoid helical bundles showed nanomolar dissociation constants for zinc, suggesting that peptoids can create well-folded structures with protein-like functions.


N

OR

O

N

OR

O

trans-amide cis-amide Figure 1-10. Rapid trans- and cis-isomerization in peptoid backbone

each amino acid in target peptide sequence by peptoid monomers with identical side-

chains,74,75 or structurally similar side-chains.76,77 In addition, since proline is the only

naturally existing N-substituted amino acid, proline in target peptide sequences can be

replaced by peptoid monomers and this strategy has yielded some exciting results

including peptoid-based SH3 ligands,78,79 somatostatin analogues,80 plant peptide

hormone analogues,81 and antimicrobial peptide analogues.82

Instead of substituting individual peptide residues with peptoid monomers,

several peptoids have been designed based on the secondary structures of the parent

peptides to mimic the structures and consequently the biological function of bioactive

peptides, including HDM2-p53 interaction inhibitors,83 lung surfactant protein

mimics,84-86 and antimicrobial peptoids87-89. To imitate glycoproteins and their

important functions in cell-cell recognition, glycopeptoids have been successfully

created, however, their biological activity remains to be tested.90

Since biological function is exquisitely affected by even minor structural

details, transforming peptides into peptoids is not straightforward. Peptoid

incorporation into a peptide

backbone causes shifting of side-

chains and removes H-bond

donors. This might result in an

altered conformation, which will

deteriorate the biological activity of peptoid analogues, because it is often crucial for

each amino acid in a peptide/protein to have precise spacing and geometry for protein-

protein interactions. The structural flexibility of peptoids due to rapid cis/trans


isomerization at amide bonds can also be problematic (Fig. 1-10). This flexible

backbone structure of peptoids can lead to a large entropy penalty upon peptoid

binding to the target protein, and possibly cause low binding affinity for peptoid

therapeutics. Nevertheless, the simplicity of the synthesis, a diverse set of

commercially readily available primary amines as monomers, and the great chemical

versatility (e.g., side-chain cyclized helical peptoids91) provide almost infinite

opportunity to explore peptoids as attractive druggable molecules. Therefore, we

believe that there is a great need for developing a generally applicable methodology to

guide the incorporation of peptoid residues into therapeutic peptides, thereby allowing

us to create better peptoid-based bioactive therapeutics.

Overview of the thesis

To address urgent needs in developing an appropriate assay system to identify

antivirals targeting viral entry, hRSV fusion protein F has been a main subject in this

thesis as a representative of class I fusion protein-mediated viral entry. We designed a

novel assay platform for screening potential hRSV entry inhibitors and then screened

possible peptides derived from the hRSV F protein and from phage displayed peptide

libraries. We also developed new peptidomimetic approaches to improve the desired

properties of therapeutic peptides.

In Chapter 2, we established and validated a protein-based fluorescence

polarization (FP) assay as a screening platform to specifically identify hRSV entry

inhibitors. We hypothesized that a 5-helix bundle (5HB), an engineered hRSV F

protein, can provide a well-defined binding site for potential antiviral candidates


targeting hRSV F and proved our system by carrying out peptide truncation studies,

suggesting C20 peptide (a 20 amino acid-long peptide) can be a potential anti-hRSV

agent with a low micromolar binding affinity. In addition, we examined small

molecule fusion inhibitors using the 5HB-based system, as the mechanism of action of

these compounds has yet to be fully understood and our assay could provide a tool to

address this. This 5HB-based FP assay was then utilized in all subsequent work.

In Chapter 3, using the C20 peptide as a parent molecule, we explored

NMEGylation as a simple method to enhance the bioavailability of relatively short

peptides, which are otherwise less attractive as biotherapeutics. The studies reported in

Chapter 4 are a part of our effort to develop a generally applicable method to convert

therapeutic peptides into peptoid-based peptidomimetics. The knowledge gained here

is used to generate structurally constrained peptomeric hRSV inhibitors with

expectation of enhanced !-helicity and thus increased binding affinity. In Chapter 5,

we expand our scope of searching anti-hRSV agents to 12-mer peptides derived from

phage-displayed peptide libraries. In addition to exploring the binding activity of the

12-mer peptides to the 5HB using our FP assays, we made an effort to structurally

examine the interaction between 12-mer peptides and the 5HB by X-ray

crystallography. Finally, the conclusions and further implications of this research are

described in Chapter 6. Consequently we believe that our results in this thesis will

provide new insights into the development of antivirals targeting hRSV fusion,

expecting that our strategy can be applied to other viruses sharing similar viral fusion

mechanism. Also, we anticipate that the method proposed here will provide a simple


and easily applicable method for the design of bioactive peptoid-based

peptidomimetics.


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43. Sia, S.K., Carr, P.A., Cochran, A.G., Malashkevich, V.N. & Kim, P.S. Short constrained peptides that inhibit HIV-1 entry. Proc Natl Acad Sci U S A 99, 14664-14669 (2002).

44. Angelo, N.G. & Arora, P.S. Solution- and solid-phase synthesis of triazole oligomers that display protein-like functionality. J Org Chem 72, 7963-7967 (2007).

45. Wiley, R.A. & Rich, D.H. Peptidomimetics derived from natural products. Med Res Rev 13, 327-384 (1993).

46. Sabatino, D., Proulx, C., Klocek, S., Bourguet, C.B., Boeglin, D., Ong, H. & Lubell, W.D. Exploring side-chain diversity by submonomer solid-phase aza-peptide synthesis. Org Lett 11, 3650-3653 (2009).

47. Tang, H., Doerksen, R.J. & Tew, G.N. Synthesis of urea oligomers and their antibacterial activity. Chem Commun (Camb), 1537-1539 (2005).

48. Wu, Y.D. & Gellman, S. Peptidomimetics. Acc Chem Res 41, 1231-1232 (2008).

49. Horne, W.S. & Gellman, S.H. Foldamers with heterogeneous backbones. Acc

Chem Res 41, 1399-1408 (2008).

50. Czyzewski, A.M. & Barron, A.E. Protein and peptide biomimicry: Gold-mining inspiration from nature’s Ingenuity. AlChE J. 54, 1 - 7 (2008).


51. Stephens, O.M., Kim, S., Welch, B.D., Hodsdon, M.E., Kay, M.S. & Schepartz, A. Inhibiting HIV fusion with a beta-peptide foldamer. J Am Chem

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52. Kritzer, J.A., Stephens, O.M., Guarracino, D.A., Reznik, S.K. & Schepartz, A. beta-Peptides as inhibitors of protein-protein interactions. Bioorg Med Chem 13, 11-16 (2005).

53. Kritzer, J.A., Lear, J.D., Hodsdon, M.E. & Schepartz, A. Helical beta-peptide inhibitors of the p53-hDM2 interaction. J Am Chem Soc 126, 9468-9469 (2004).

54. Seebach, D., Beck, A.K. & Bierbaum, D.J. The world of beta- and gamma-peptides comprised of homologated proteinogenic amino acids and other components. Chem. Biodivers. 1, 1111-1239 (2001).

55. Seebach, D., Hook, D.F. & Glattli, A. Helices and other secondary structures of beta- and gamma-peptides. Biopolymers 84, 23-37 (2006).

56. Simon, R.J., Kania, R.S., Zuckermann, R.N., Huebner, V.D., Jewell, D.A., Banville, S., Ng, S., Wang, L., Rosenberg, S., Marlowe, C.K. & et al. Peptoids: a modular approach to drug discovery. Proc Natl Acad Sci U S A 89, 9367-9371 (1992).

57. Fowler, S.A., Luechapanichkul, R. & Blackwell, H.E. Synthesis and characterization of nitroaromatic peptoids: fine tuning peptoid secondary structure through monomer position and functionality. J Org Chem 74, 1440-1449 (2009).

58. Zuckermann, R.N. & Kodadek, T. Peptoids as potential therapeutics. Curr

Opin Mol Ther 11, 299-307 (2009).

59. Kirshenbaum, K., Barron, A.E., Goldsmith, R.A., Armand, P., Bradley, E.K., Truong, K.T., Dill, K.A., Cohen, F.E. & Zuckermann, R.N. Sequence-specific polypeptoids: a diverse family of heteropolymers with stable secondary structure. Proc Natl Acad Sci U S A 95, 4303-4308 (1998).


60. Patch, J.A., Kirshenbaum, K., Seurynck-Servoss, S.L. & Zuckermann, R.N. Versatile Oligo(N-Substituted) Glycines: The Many Roles of Peptoids in Drug

Discovery (Wiley-VCH, Weinheim, 2004).

61. Zuckermann, R.N., Kerr, J.M., Kent, S.B.H. & Moos, W.H. Efficient method for the preparation of peptoids [Oligo(N-substituted glycines)] by submonomer solid-phase synthesis. J Am Chem Soc 114, 10646-10647 (1992).

62. Burkoth, T.S., Fafarman, A.T., Charych, D.H., Connolly, M.D. & Zuckermann, R.N. Incorporation of unprotected heterocyclic side chains into peptoid oligomers via solid-phase submonomer synthesis. J Am Chem Soc 125, 8841-8845 (2003).

63. Gorske, B.C., Stringer, J.R., Bastian, B.L., Fowler, S.A. & Blackwell, H.E. New strategies for the design of folded peptoids revealed by a survey of noncovalent interactions in model systems. J Am Chem Soc 131, 16555-16567 (2009).

64. Huang, K., Wu, C.W., Sanborn, T.J., Patch, J.A., Kirshenbaum, K., Zuckermann, R.N., Barron, A.E. & Radhakrishnan, I. A threaded loop conformation adopted by a family of peptoid nonamers. J Am Chem Soc 128, 1733-1738 (2006).

65. Pokorski, J.K., Jenkins, L.M., Feng, H., Durell, S.R., Bai, Y. & Appella, D.H. Introduction of a triazole amino acid into a peptoid oligomer induces turn formation in aqueous solution. Org Lett 9, 2381-2383 (2007).

66. Wu, C.W., Kirshenbaum, K., Sanborn, T.J., Patch, J.A., Huang, K., Dill, K.A., Zuckermann, R.N. & Barron, A.E. Structural and spectroscopic studies of peptoid oligomers with alpha-chiral aliphatic side chains. J Am Chem Soc 125, 13525-13530 (2003).

67. Armand, P., Kirshenbaum, K., Goldsmith, R.A., Farr-Jones, S., Barron, A.E., Truong, K.T., Dill, K.A., Mierke, D.F., Cohen, F.E., Zuckermann, R.N. & Bradley, E.K. NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains. Proc Natl Acad Sci U S A 95, 4309-4314 (1998).

68. Wu, C.W., Seurynck, S.L., Lee, K.Y. & Barron, A.E. Helical peptoid mimics of lung surfactant protein C. Chem Biol 10, 1057-1063 (2003).


69. Jang, H., Fafarman, A., Holub, J.M. & Kirshenbaum, K. Click to fit: versatile polyvalent display on a peptidomimetic scaffold. Org Lett 7, 1951-1954 (2005).

70. Shin, S.B., Yoo, B., Todaro, L.J. & Kirshenbaum, K. Cyclic peptoids. J Am

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71. Burkoth, T.S., Beausoleil, E., Kaur, S., Tang, D., Cohen, F.E. & Zuckermann, R.N. Toward the synthesis of artificial proteins: the discovery of an amphiphilic helical peptoid assembly. Chem Biol 9, 647-654 (2002).

72. Lee, B.C., Zuckermann, R.N. & Dill, K.A. Folding a nonbiological polymer into a compact multihelical structure. J Am Chem Soc 127, 10999-11009 (2005).

73. Lee, B.C., Chu, T.K., Dill, K.A. & Zuckermann, R.N. Biomimetic nanostructures: creating a high-affinity zinc-binding site in a folded nonbiological polymer. J Am Chem Soc 130, 8847-8855 (2008).

74. Ruijtenbeek, R., Kruijtzer, J.A., van de Wiel, W., Fischer, M.J., Fluck, M., Redegeld, F.A., Liskamp, R.M. & Nijkamp, F.P. Peptoid - peptide hybrids that bind Syk SH2 domains involved in signal transduction. Chembiochem 2, 171-179 (2001).

75. Caporale, A., Schievano, E. & Peggion, E. Peptide-peptoid hybrids based on (1-11)-parathyroid hormone analogs. J Pept Sci 16, 480-485 (2010).

76. Hoffmann, B., Ast, T., Polakowski, T., Reineke, U. & Volkmer, R. Transformation of a biologically active Peptide into peptoid analogs while retaining biological activity. Protein Pept Lett 13, 829-833 (2006).

77. Zimmermann, J., Kuhne, R., Volkmer-Engert, R., Jarchau, T., Walter, U., Oschkinat, H. & Ball, L.J. Design of N-substituted peptomer ligands for EVH1 domains. J Biol Chem 278, 36810-36818 (2003).

78. Nguyen, J.T., Turck, C.W., Cohen, F.E., Zuckermann, R.N. & Lim, W.A. Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors. Science 282, 2088-2092 (1998).


79. Nguyen, J.T., Porter, M., Amoui, M., Miller, W.T., Zuckermann, R.N. & Lim, W.A. Improving SH3 domain ligand selectivity using a non-natural scaffold. Chem Biol 7, 463-473 (2000).

80. Mattern, R.H., Moore, S.B., Tran, T.A., Rueter, J.K. & Goodman, M. Synthesis, biological activities, and conformational sutdies of somatostatin analogues. Tetrahedron 56, 9819-9831 (2000).

81. Kondo, T., Yokomine, K., Nakagawa, A. & Sakagami, Y. Analogs of the CLV3 Peptide: Synthesis and Structure-Activity Relationships Focused on Proline Residues. Plant Cell Physiol (2010).

82. Zhu, W.L., Lan, H., Park, Y., Yang, S.T., Kim, J.I., Park, I.S., You, H.J., Lee, J.S., Park, Y.S., Kim, Y., Hahm, K.S. & Shin, S.Y. Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide. Biochemistry 45, 13007-13017 (2006).

83. Hara, T., Durell, S.R., Myers, M.C. & Appella, D.H. Probing the structural requirements of peptoids that inhibit HDM2-p53 interactions. J Am Chem Soc 128, 1995-2004 (2006).

84. Seurynck-Servoss, S.L., Dohm, M.T. & Barron, A.E. Effects of including an N-terminal insertion region and arginine-mimetic side chains in helical peptoid analogues of lung surfactant protein B. Biochemistry 45, 11809-11818 (2006).

85. Brown, N.J., Johansson, J. & Barron, A.E. Biomimicry of surfactant protein C. Acc Chem Res 41, 1409-1417 (2008).

86. Brown, N.J., Wu, C.W., Seurynck-Servoss, S.L. & Barron, A.E. Effects of hydrophobic helix length and side chain chemistry on biomimicry in peptoid analogues of SP-C. Biochemistry 47, 1808-1818 (2008).

87. Patch, J.A. & Barron, A.E. Helical peptoid mimics of magainin-2 amide. J Am

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89. Chongsiriwatana, N.P., Patch, J.A., Czyzewski, A.M., Dohm, M.T., Ivankin, A., Gidalevitz, D., Zuckermann, R.N. & Barron, A.E. Peptoids that mimic the structure, function, and mechanism of helical antimicrobial peptides. Proc Natl

Acad Sci U S A 105, 2794-2799 (2008).

90. Seo, J., Michaelian, N., Owens, S.C., Dashner, S.T., Wong, A.J., Barron, A.E. & Carrasco, M.R. Chemoselective and microwave-assisted synthesis of glycopeptoids. Org Lett 11, 5210-5213 (2009).

91. Vaz, B. & Brunsveld, L. Stable helical peptoids via covalent side chain to side chain cyclization. Org Biomol Chem 6, 2988-2994 (2008).

27

Chapter Two

Design and evaluation of a structure-guided screening platform for

peptide-based hRSV entry inhibitors!

Human Respiratory Syncytial Virus (hRSV)

hRSV is a negative-sense single-stranded RNA virus and belongs to the

Pneumovirus genus of the Paramyxoviridae family. Among its many glycoproteins,

which are critical in viral functions including reproduction, the attachment protein (G)

and the fusion protein (F) are major players in the infection and pathogenesis of

hRSV, and are thus relatively well studied. The G protein is known to be required for

attachment of the virus to respiratory epithelial cells, whereas the F protein directly

mediates the viral fusion and entry, inducing the formation of the characteristic

syncytium, that is a large multi-nucleated mass of cytoplasm.1

The F protein is synthesized as a precursor F0, and is then processed by

proteolytic cleavage, subsequently yielding two subunits, F1 and F2. This step is

! This chapter is adapted from the following publication:

Park, M. et al. (2011) “A fluorescence polarization assay using an engineered hRSV F protein as a

direct screening platform” Anaytical Biochemistry 409, 2, 195-201

Figure 2-1. Schematic diagram of hRSV fusion (F) protein F1 and F2 are formed after proteolytic cleavage (arrow) of the precursor protein (F0) by furine-like protease. The fusion peptide (FP) and transmembrane domain (TM) are indicated. Adjacent to FP and TM, heptad repeat regions, HRA and HRB are shown.

Chapter Two: Design and evaluation of a structure-guided screening platform for peptide-based

hRSV entry inhibitors

28

required for activation.2 As a strong indication of its importance, the sequence of the F

protein is highly conserved among Paramyxoviridae family. As shown in Fig. 2-1,

two hydrophobic heptad repeats (HRA and HRB) are located adjacent to the fusion

peptide (FP) and transmembrane domain (TM) with approximately 250 residues of

intervening sequence between them. The HRA and HRB domains contain a sequence

motif suggesting "-helical coiled-coil structure.

Viral entry into the host cells is an early but crucial event to the hRSV

infectious cycle. Upon attachment via the protein G, hRSV F protein facilitates

membrane fusion with the host cells at neutral pH, similar to other paramyxoviruses

such as measles virus. A more detailed mechanism has been proposed based on pre-

and post-fusion structures of the F protein as discussed earlier in Chapter 1.3

(A) (B)

Figure 2-2. X-ray crystal structure of hRSV 6HB (A) a side and (B) top view of the hRSV F 6HB are shown. HRA and HRB are color-coded in green and blue, respectively. HRA helices form a trimeric innercore and HRB helices pack against the innercore in anti-parallel manner, completing the 6HB assembly. Figures were generated by Pymol software.



29

The hRSV F protein-mediated membrane merger is particularly interesting,

because it occurs at the cell surface, so the process is accessible to inhibition by

antivirals without requiring cell permeability. It thus has been recognized as an

attractive therapeutic target, yielding numerous peptidic or small molecule fusion

inhibitors.4-16 In addition, X-ray structural studies have shown that the HRB regions

pack in hydrophobic grooves formed on the surface of the HRA trimer core (Fig. 2-2)

showing striking similarity to HIV-1 gp41 core structure (Fig. 1-2).17 In the case of

HIV-1 gp41, synthetic peptides corresponding to HRA or HRB can effectively inhibit

HIV infection.18,19 Fuzeon® (enfuvirtide, Roche), a drug currently approved in the

U.S. for use against HIV infections, uses this mechanism of inhibition. Collectively,

all these features strongly support the hypothesis that targeting the F protein and the

viral fusion process would be effective in identifying hRSV entry inhibitors.

Urgent need to develop hRSV treatment

Despite attempts to develop safe, cost-effective treatments to control hRSV-

associated illness,4,5 hRSV remains the leading pathogen causing lower respiratory

tract infections mainly in infants and young children, and a severe public health

problem worldwide. hRSV infections cause more than 140,000 pediatric

hospitalizations20 and 2,000 deaths in the United States alone with costs of $356 – 585

million annually.1,21-24 Most children are infected with hRSV at least once before the

age of 2, and recurrence is very common.25 hRSV has also been an increasingly

recognized cause of high morbidity and mortality in immunosuppressed patients and

the elderly. In case of the elderly, hRSV infections have been reported as significant



30

cause for many complications such as pneumonia, resulting in up to 10,000 deaths

annually.26,27 However, there are no treatment options available to specifically treat

hRSV infections and related complications except for supportive care such as oxygen

supply.

Current hRSV treatment options

Antiviral drug discovery to control hRSV infections has mainly relied on the

screening of chemical libraries or natural products using common virology assays and

animal models, yielding limited success.5,28,29 To date, a nucleoside analog, Ribavirin,

is the only clinically approved antiviral agent to treat hRSV infections by interfering

with the RNA metabolism required for viral replication. However, due to its

controversial efficacy, potential cytotoxicity and severe side effects, its clinical usage

is tightly restricted. As discussed earlier in this chapter, the membrane merger between

virus and the host cells is the key step that enables the viral genome to enter and

initiate the infectious cycle of hRSV. Because this event happens extracellularly, it is a

very attractive therapeutic target to develop antivirals against hRSV infections,

resulting in many small molecule drug candidates including BTA9881, BMS433771,

TMC353121, VP14637, and RFI641 (see small molecule fusion inhibitors section

below). These candidates were identified with tissue-cell culture based assays and

their mechanism of action is poorly understood except that the compounds presumably

interrupt the formation of 6HB during viral fusion by binding in the hydrophobic

pocket (Fig. 2-3). However, these efforts have resulted in a high failure rate and none

are in advanced stages of clinical trails.30,31 With treatment options limited to measures



31

such as supportive care, development of safe and specific agents against hRSV is

essential.32

Current options for hRSV prevention

Because of the limited treatment options available, prevention strategies are

highly desirable.20,29 However, vaccine development faces several obstacles: the need

to immunize very young infants who may not respond adequately to vaccination and

the existence of antigenically different hRSV strains (A and B).29 A humanized

monoclonal antibody, Synagis# (palivizumab, MedImmune) targeting a conserved

neutralizing epitope on the hRSV F protein successfully inhibits viral fusion and was

FDA-approved in 1998. Since then, it has been used essentially as the only option for

(A) (B)

Figure 2-3. A key interaction between HRA and HRB helices and the hydrophobic pocket formed by neighboring HRA helices in the 6HB assembly (A) Two phenylalanine residues of the hRSV F HRB domain marked in red play a crucial role in the interaction with the hRSV F HRA helices shown in green by packing into (B) the hydrophobic pocket shown in red dotted are formed by HRA helices. Figures were generated by PyMol software.



32

hRSV prevention efforts; however, its usage is restricted to high-risk children (babies

born at less than 36 weeks or who have heart or lung problems). Because Synagis#

needs to be administered throughout the flu outbreak season (November to March), the

cost has become a limiting factor for those who want the treatment.

Numax$ (Motavizumab, Medi-524, MedImmune) is a 2nd generation

humanized monoclonal antibody. It is directly derived from Synagis# with only a 13

amino acid difference, but is over 20-fold more potent than Synagis# in in vitro

microneutralization assays.32 However, as of September 20, 2010, its FDA approval is

on hold and the FDA has requested more data from an additional trial of the drug to

support a satisfactory risk/benefit profile for the prophylaxis indication.33

Development of screening methods for hRSV fusion inhibitors

To our knowledge, there is no established, simple, non-cell-based method to

screen potential antivirals specifically targeting the hRSV F protein. Previously, the 5-

Helix of the HIV-1 fusion protein gp41 was shown to be a viral entry inhibitor,34 as

well as a suitable target for screening small molecule libraries in a high-throughput

format.35 This suggests that similar approaches would be applicable to other viruses,

like hRSV, that rely on class I viral fusion proteins. We thus created a 5-Helix Bundle

(5HB), variant hRSV F protein, and developed a competitive fluorescence polarization

(FP) based assay using the 5HB as a target protein and a fluorescently labeled peptide

as a tracer. To validate that the competitive FP-based 5HB assay can provide a reliable

screening platform, a series of N- and C- terminally truncated peptides derived from

HRB domain of the hRSV F protein were synthesized and tested. Thus, we



33

demonstrated a simple, fast, and low-cost in vitro fluorescence polarization assay that

can be readily applied to libraries of peptides, peptidomimetics, or small molecules to

rapidly screen potential hRSV fusion inhibitors (Scheme 2-1).

Scheme 2-1. Strategy designed and developed in this study to identify hRSV fusion inhibitors targeting specifically hRSV F protein. In the right panel (shaded), the 5HB serves as a screening platform for identifying potential hRSV fusion inhibitors. Once inhibitor candidates are selected, these inhibitors can be further studied in tissue-cell culture-based assay. Ideally we expect that the inhibitor candidates identified from the 5HB-based assay will prevent the 6HB formation of hRSV F, thereby blocking viral fusion and entry.



34

5-Helix Bundle construct design, expression, purification and secondary structure

analysis

A 5-Helix construct of HIV-1 fusion protein gp41 has been tested as a fusion

inhibitor34 and used as a target protein for screening small molecule libraries.35

However, analogous constructs for hRSV F have only been tested as fusion inhibitors

or vaccine candidates.16,36 Therefore, we designed a 5-Helix Bundle (5HB) construct

to specifically mimic the 6HB that forms during hRSV infection of the host cell. The

5HB was generated by connecting three HRA and two HRB helices in an alternating

sequence using short peptide linkers (Fig. 2-4A). The absence of the 3rd HRB helix in

the 5HB would create a large open binding site for potential fusion inhibitors. Soluble

5HB was expressed in BL21 (DE3) cells and purified by metal-affinity

chromatography (Fig. 2-4B).

(A) (B)

Figure 2-4. An illustration of hRSV 5-Helix Bundle (5HB) with a resulting SDS-PAGE of 5HB purification (A) the designed 5HB and its single-chain polypeptide sequence are shown. A binding site for the missing 3

rd HRB and potential fusion

inhibitors is shown in dotted line. (B) Purified 5HB samples were analyzed by SDS-PAGE on a 12% non-reducing gel (M; molecular marker, FT; flow through, W; wash, and E; elution fractions).



35

Since the 5HB is composed of 5 difference helices, it is very crucial to confirm

that this protein construct is well-folded. Therefore, the secondary structure of the

5HB was assessed by Circular Dichroism (CD) spectroscopy (Fig. 2-5A). As

expected, the 5HB presented very intense "-helical features, showing two strong

negative peaks at 208 and 220 nm along with a strong positive peak at around 190 nm.

The helical content was calculated using Dichroweb,37,38 resulting in approximately

90% "-helicity. The thermal stability of the 5HB was also examined in the

temperature range from 0 to 85 °C and we did not observe any sign of thermal

denaturation of the 5HB, indicating that the secondary structure of the 5HB is highly

stable (Fig. 2-5B). This is consistent with observation for the HIV-1 gp41 5-Helix.34

(A) (B)

Figure 2-5. Secondary structure analysis and thermal stability of 5HB by CD spectroscopy (A) CD spectrum of 5HB in 10 mM PBS at pH 7.4. (B) The melting curve of

5HB obtained from the ellipticity measurements at 222 nm between 0 and 85 °C. ([!]: per

residue molar ellipticity measured in degrees % cm2 % dmol

-1 % residue

-1)



36

(A)

Figure 2-6. A schematic diagram of Trx-C49 with a resulting SDS-PAGE of purified Trx-C49 (A) An illustration of the designed Trx-C49 and its single-chain polypeptide sequence are shown (B) Purified Trx-C49 samples were analyzed by SDS-PAGE on a 12% non-reducing gel (M; molecular marker, and E; elution fractions). Caluated molecular weigt of Trx-C49 is 24587.5 Da.

(B)

Validation of 5HB as an hRSV F protein 6HB mimic by ELISA assays

To validate the functionality of the 5HB as a screening platform, we carried

out ELISA assays on the 5HB using a previously described procedure.39,40 To do so, a

full length of C-peptide (C49) derived from hRSV F HRB domain containing 49 amino

acids was prepared (Fig. 2-6). Because this peptide is the missing outer helix, it should

bind to the 5HB tightly forming a stable 6HB, and thus proving that the 5HB provides

a high affinity binding site as anticipated. Since the C49 was too short to express in

E.coli, it was expressed as a thioredoxin (Trx) fusion protein (Trx-C49). Binding of

Trx-C49 to the 5HB was monitored with increasing concentration of the 5HB. To

monitor the binding of Trx-C49 to the 5HB quantitatively, an ELISA assay against the

S-tag on the Trx-C49 was performed, confirming that Trx-C49 tightly binds to the 5HB

with a nanomolar affinity (Kd = 13.7 nM) (Fig. 2-7A).



37

Saturation binding FP measurement

Fluorescence polarization (FP) has been widely used for a direct, nearly

instantaneous spectroscopic measurement of molecular interactions such as protein-

protein41-43, DNA-protein44, and small molecule-protein45,46 interactions. FP is also

more straightforward (i.e., fewer steps) and less expensive (e.g., no antibodies) than

other methods such as ELISA. To develop a reliable FP assay, the binding affinity as

well as specificity of the probe to the target protein should be high.47 Previously, it has

been shown that a series of 35-amino acid-long peptides from the conserved HRB

domain within hRSV F protein could block syncytium formation with EC50 values in

the range of 0.015 – 0.25 µM.6 Therefore, we decided to use T-108 (35mer:

YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL) as a probe for developing a FP

assay for the following reasons: First, the low EC50 value of 0.051 µM suggests that T-

(A) (B)

Figure 2-7. Indirect and competitive ELISA assays (A) The binding of Trx-C49 to the 5HB was observed as the concentration of Trx-C49 increased. (B) The competitive capability of Fl-C35 over Trx-C49 was tested using 7.8 nM of the 5HB, 4 nM of Trx-C49 and

Fl-C35 concentrations ranging from 0 to 10 µM.



38

108 should bind tightly to the 5HB; second, T-108 contains two phenylalanine

residues (F483 and F488) located at the N-terminus of the HRB region, which engage a

deep hydrophobic pocket located at the C-terminus of the HRA helices, that is

believed to be a good antiviral drug target (Fig. 2-3).10,11,48 This T-108 peptide was

labeled with fluorescein at its N-terminus (Fl-C35) to sever as a probe in FP.

To validate this peptide as a probe, we need to demonstrate that it can

effectively compete with the 6th helix of the F protein, as well as tightly and stably

bind the 5HB at low concentration. Using the ELISA test with Trx-C49, we were able

to show that Fl-C35 could displace the 6th helix from the 5HB construct with a

comparative IC50 value of ~ 40 nM (Fig. 2-7B). To determine the binding affinity of

Fl-C35, we used a fixed concentration of 5 nM Fl-C35 and monitored the FP response

(A) (B)

Figure 2-8. Binding titration curve of Fl-C35 and its binding stability to the 5HB The fluorescence polarization response of Fl-C35 binding to the 5HB was monitored as the concentration of the 5HB increased. The experiment was performed using 5 nM Fl-C35 and the 5HB concentration ranged from 0 to 500 nM. (B) The stability of Fl-C35 binding to the 5HB was monitored over a 24 hr period using 5 nM of Fl-C35 in the presence of increasing amount of the 5HB.



39

of Fl-C35 with increasing concentrations of the 5HB, as shown in Fig. 2-8A. The FP

results were consistent with high affinity binding (Kd = 21 nM). The stability of the FP

assay using Fl-C35 and the 5HB is also important for its potential use for a high-

throughput screening format. We therefore tested the stability of Fl-C35 binding to the

5HB by incubating the plate at room temperature over 24 hrs (Fig. 2-8B). The

resulting binding curves show that this assay is highly stable and robust, which will

allow us to carry out large-scale tests at room temperature.

Specificity of Fl-C35 binding to the 5HB

The specificity of Fl-C35 binding to the 5HB was cross-tested against a system

derived from the Epstein-Barr virus (EBV), a member of the human herpesviruses.

EBV requires a number of envelope glycoproteins for membrane fusion with the host

cells. Particularly, the interaction of the EBV gp42 and the gH/gL complex are crucial

in viral entry into B cells. The Jardetzky laboratory has reported that the EBV gp42-

derived FITC-30mer specifically binds to EBV gH/gL protein with a low nanomolar

Kd using a FP assay.49,50 To confirm the specificity of both the hRSV 5HB and the Fl-

C35 probe, we tested the EBV gp42-derived FITC-30mer against the hRSV 5HB (Fig.

2-9A) and our hRSV-derived Fl-C35 against the EBV gH/gL complex under the same

conditions (Fig. 2-9B). There was no evidence of nonspecific binding in these

controls, indicating the interaction between Fl-C35 and the 5HB is specific and can

provide a solid basis for developing a competitive FP-based 5HB assay.



40

Competitive FP assays

Based on the Kd value observed in the saturation binding FP assay, we

established a competitive FP assay to evaluate potential inhibitors based on their

ability to displace the Fl-C35 (probe) from the 5HB (target). We first tested unlabelled

C35 against Fl-C35 in the presence of the 5HB (Fig. 2-10A). Unlabelled C35 peptide

blocked the increase in polarization value with an IC50 of 38 nM, competing with Fl-

C35 over the binding site on the 5HB. To demonstrate the effectiveness of the 5HB-

screening platform as a tool capable of evaluating molecules with different binding

affinities, we investigated a series of short peptides. Manufacturing longer bioactive

peptides can be problematic due to high cost, but shorter unstructured peptides can

easily lose their efficacy, and these contradictory criteria greatly affect for the design

of peptide therapeutics.

(A) (B)

Figure 2-9. Specificity and selectivity of Fl-C35 to the 5HB (A) 5 nM of Fl-C35 in the presence of increasing amount of the Epstein-Barr virus (EBV) gH/gL, a fusion protein that leads the EBV infection was tested. (B) 5 nM of EBV gp42 FITC-30mer with a wide range of the 5HB in concentration was monitored.



41

Table 2-1. Peptides derived from the HRB domain of hRSV F protein and their

sequences and binding affinities to the 5HB

ID Sequence (amino to carboxy) %

Inh.a

IC50

(µM)

HRBb NFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN - -

T-108c YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL 0.051

C35 YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL 100 0.038

C30 VFPSDEFDASISQVNEKINQSLAFIRKSDE 100 6.80

C20 ISQVNEKINQSLAFIRKSDE > 90 14.92

C17 VNEKINQSLAFIRKSDE < 50 > 100

C13 INQSLAFIRKSDE NMd > 500

C10 SLAFIRKSDE NMd > 500

N15 VFPSDEFDASISQVN < 50 > 100

a % Inhibition of each peptide at the concentration of 100 µM was calculated.

b HRB sequence from hRSV F protein

c For T-108, the reported value of EC50 from Lambert et al. is used instead of an IC50 value, crude

peptide T-108 was analyzed for its ability to prevent cytopathologic effect (CPE) in infectivity assays

with hRSV.

d NM: Not measurable

We therefore prepared a series of truncated peptides derived from the HRB

domain of hRSV F protein (Table 2-1) and investigated their ability to compete

against Fl-C35 using our competitive 5HB-based FP assay. The truncated peptides

tested in this study mostly do not have the two phenylalanines that bind to the

hydrophobic pocket (Fig. 2-3) that has been the focus of small molecule drug

discovery effort.4,48,51 However, in previous work by Lambert et al., multiple 35-mer

peptides that lacked those phenylalanines still exhibited significant antiviral activity.6

Notably, our hope was that shorter peptides with even modest binding affinity could

act synergistically with small molecule drugs that would target the hydrophobic



42

binding pocket. The range of truncation peptide length described here addresses

intermediate peptide lengths (< 35 amino acid-long), so we believe our study can

provide an evidence that the entire hydrophobic groove on the C-terminus of the

neighboring HRA helices would be available as a potential drug target for developing

specific hRSV fusion inhibitors. The results summarized in Fig. 2-10B and Table 2-1

provide quantitative observations of the binding activity of shorter peptides (< 35

amino acid-long).

Suitability as a high-throughput screening system

Previous pharmaceutical lead compounds were identified using week-long

tissue cell cultures using live virus. Since the FP assay system could reduce costs by at

least an order of magnitude, it could enable much broader efforts to develop effective

(A) (B)

Figure 2-10. Competitive FP assays of unlabeled C35 and N- and C-terminally truncated peptides (A) Unlabeled C35 were competed with 5 nM Fl-C35 in the presence of 20 nM 5HB and its ability to displace Fl-C35 was monitored by FP. (B) Various concentrations of peptides were competed with 5 nM Fl-C35 in the presence of 20 nM 5HB.



43

hRSV treatments. As discussed above, the 5HB-baed FP assays are stable at room

temperature over 24 hrs (Fig. 2-8B). In addition, the reproducibility of measurements

for free and unbound Fl-C35 controls were examined (Fig. 2-11) for calculating the Z’

factor, a measure of the quality and robustness of an assay without test compounds.47

The Z’ factor was determined based on the guidelines (Equation 2-1) provided by

National Institutes of Health (NIH),52 resulting in Z’ factor = 0.8, which suggests our

assay can be directly applied to the HTS format.

Figure 2-11. Assay robustness test by FP measurements of free and bound Fl-C35 controls

!

" Z =1#3($p + $n)

µp#µn

Equation 2-1. Equation for calculating Z’ factor Values of Z’ between 0.7 – 0.9 correspond to a good assay while a Z’ value of 0.5 corresponds to the minimum acceptable value for the HTS. Means and standard deviations of both positive (p) and negative (n)

controls (µp, &p, and µn, &b)



44

Small molecule fusion inhibitors tested in 5HB-based competitive FP assays

As discussed earlier, since the hydrophobic cavity was identified as a

potentially attractive drug target, several small molecules that prevent hRSV infection

in cell studies have been reported (Table 2-2 and Fig. 1-12).48

Table 2-2. Human respiratory syncytial virus fusion inhibitors

Drug candidates Type of compounds Development status Administration Ref.

TMC353121 Viral fusion inhibitor Preclinical, ongoing Inhalation or

oral delivery 53,54

BMS433771 Viral fusion inhibitor Discontinued Oral delivery 10,55,56

RFI641 Viral fusion inhibitor Discontinued Inhalation 57

VP14637 Viral fusion and

replication inhibitor Discontinued Inhalation 9,58,59

BTA9881 Viral fusion inhibitor Insufficient safety in Phase I Oral delivery 60

O

NHN N

NHN

N

HO

OH

(1) TMC353121

(4) BMS433771

NN

NN

HO

O

N

(5) Biota

NN

N

Cl FO

HN N

N

N

HO

(2) JNJ240868

NH2N

N

HN

(3) Trimeris

N

N NN

O

Figure 2-12. Structures of hRSV fusion inhibitors tested in this study



45

Figure 2-13. Competitive FP assays of small molecule fusion inhibitors Unlabeled C35

was at 10 µM used to show 100% inhibition of 5HB·Fl-C35 bound. As a negative control

(0% inhibition), %5HB·Fl-C35 bound was measured in the absence of inhibitors (no cmpd).

Each compound at 0, 10, and 100 µM was tested in the presence of 5 nM Fl-C35 and 20

nM 5HB. (Cmpd; compound, cmpd1; TMC353121, cmpd2; JNJ240868, cmpd3; Trimeris, cmpd4; BMS433771, and cmpd 5; Biota)

Even though the mechanisms of the antiviral activity of these compounds are

poorly understood, it is predominantly believed that the small molecules bind to the

hydrophobic cavity on the inner core of HRA helices, thereby preventing the 6HB

formation and consequently blocking viral fusion. Recently, it was suggested that a

small molecule inhibitor of hRSV F entry may act not by blocking the 6HB formation,

but by distorting the final 6HB conformation.53 This study indicates that the inhibitor

(TMC353121) engages both HRA and HRB regions and may thereby enhance the

6HB assembly into a non-functional structure.

Since the Fl-C35 probe in our FP assay covers the hydrophobic pocket, we can

determine whether the small molecules bind the hydrophobic pocket (and displace the



46

probe) or bind elsewhere (and do not disturb the probe). Therefore, we examined

several hRSV F fusion inhibitors (Fig. 2-12) using the 5HB-based FP assay (Fig. 2-

13). Surprisingly, none of small molecule inhibitors showed an inhibitory effect on the

binding of Fl-C35 to the 5HB, indicating these compounds do not prevent the 6HB

formation by binding to the hydrophobic pocket. We still do not fully understand how

these compounds including TMC353121 can block viral fusion without blocking the

6HB formation. However, we believe that in addition to its role as a drug discovery-

screening tool our 5HB-based FP assay could serve as a mechanism evaluation tool for

these and other compounds. Notably, the use of an alternate probe, such as a 35-mer

that does not cover the hydrophobic pocket, could enable further in-depth mechanistic

studies. In any case, we anticipate that the 5HB system will provide a new rapid

screening platform to specifically identify small molecules that prevent hRSV F 6HB

formation.

Conclusions and future prospects

In this study, we demonstrated that a protein-based assay can be used as a direct

screening platform for identifying potential antivirals against hRSV using fluorescence

polarization (FP), which is a well-proven tool for direct and rapid measurement of

molecular interactions. Our competitive FP assay using the 5HB for mimicking the

formation of the hRSV 6HB can measure biological activities of short hRSV F-

derived peptides over a wide range of binding affinity, suggesting that this system is

sufficiently sensitive to screen weak binders to the 5HB as potential antiviral

candidates that could target not only the hydrophobic pockets but also the groove



47

formed by two neighboring HRA helices. Moreover, this assay could be suitable for an

initial high throughput screening effort to prioritize inhibitor candidates prior to tissue

culture-based assays. Since current screening strategies require week-long cell-based

assays using live virus, our rapid protein-based assay is significantly lower in cost and

can enable more comprehensive drug discovery efforts at a given level of

expenditures. This 5HB construct from the hRSV F protein may additionally prove

useful for other viruses within the paramyxovirus family for future drug discovery

efforts. Recent tests of small molecule fusion inhibitors using our 5HB-based FP assay

suggest that this 5HB assay may also allow us to specifically screen small molecules

that block or distort the 6HB formation of hRSV F.



48

Materials and Methods

Protein synthesis

5-Helix Bundle cloning: The 5HB DNA construct is composed of three N57 and two

C49 helices, representing residues 126 to 182 (HRA) and 476 to 524 (HRB) of the

hRSV F protein, respectively. Each fragment was amplified by PCR using the hRSV

strain A2 genome as a template and connected using short linkers: N57 was joined

using a linker (PPPELGGP) to C49 to generate a heterodimer, N57-C49; two

heterodimers were connected with a short linker (KGSSK); the final N57 was linked

after the second C49 via the linker (KGSSK) (Fig. 2-4A). The engineered gene

encoding 5HB was cloned between the Nde I and BamH III restriction sites of the

hexahistidine expression vector pET-15b (Novagen, San Diego, CA, USA). The

resulting plasmid carrying the complete 5HB construct was transformed into E.coli

strain BL21 (DE3) for protein expression.

5-Helix Bundle expression and purification: Protein was recombinantly expressed in

E.coli strain BL21 (DE3) grown to an OD of 0.8 at 600 nm at 37 °C in Luria-Bertani

(LB) medium. Protein expression then was induced with 0.5 mM isopropyl-'-D-

thiogalctopyranoside (ITPG), and cells were grown for an additional 20 hrs at 20 °C,

to enhance the solubility of protein.61 The cells were harvested by centrifugation at

4500 x g for 15 minutes, and the resulting cell pellet was resuspended in 20 mM

phosphate buffered saline (PBS) and stored at -80 °C. Cells were lysed in lysis buffer

(CelLytic(B cell lysis reagent [cat. no. C8740, Sigma Aldrich, Milwaukee, WI,

USA], 20 mM PBS at pH 7.4, 1 mM phenylmethylsulphonyl fluoride [PMSF],



49

protease inhibitor cocktail [Sigma Aldrich, Milwaukee, WI, USA], 1% Triton X-100,

500 mM NaCl and 0.2 mg/ml lysozyme) and incubated for 1 hr at room temperature.

Cell lysate was then clarified by centrifugation at 18,000 x g for 30 min. The soluble

fraction was immediately incubated with a Nickel-immobilized chelating sepharose

fast flow resin (cat. no. 17-0575-02, GE Healthcare, Piscataway, NJ, USA) at room

temperature for 30 min with a gentle agitation. The protein-bound resin was washed

out with more than 10 column volumes (CV) of a wash buffer (20 mM PBS at pH 7.4,

100 mM imidazole, 1% Triton X-100 and 500 mM NaCl). The 5HB was eluted with

an elution buffer (20 mM PBS at pH 7.4, 300 mM imidazole and 500 mM NaCl). The

purity of protein was assessed by SDS-PAGE and the protein was used without further

purification. Protein concentration was determined by using the BCA protein Assay

(cat. no. 23225, Pierce, Rockford, IL, USA). The final yield of soluble 5HB was

approximately 1 mg/L of cell culture with batch-to-batch variation.

Trx-C49 cloning, expression and purification: The gene sequence referred to as rec-

Trx-C49 was obtained by PCR and contains residues 476-524 of the HRB domain (Fig.

2-6A). The constructed gene was cloned into expression vector pET-32a at the

HindIII-XhoI restriction site. The resulting plasmid tagged by Thioredoxin was

transformed into E.coli BL21 (DE3) for protein expression. Thioredoxin is known to

enhance the solubility and allow the high level production of small peptides.62 The

cells were grown in LB media to an optical density (at 600 nm) of 0.8 before induction

with IPTG (1.0 mM) for 3 hrs at 37 °C. Bacterial cells were harvested and then

resuspended in PBS and subsequently frozen at –80 °C until use. Thawed



50

resuspensions were lysed with addition of lysis buffer and centrifuged to separate the

soluble fraction from insoluble fraction. The soluble protein was incubated with a

Talon resin (Clontech, Mountain View, CA, USA) at room temperature for 1 hr.

Protein was eluted in 20 mM PBS at pH 7.4 containing 500 mM NaCl, and 100 mM

imidazole. The purity of protein was judged by SDS-PAGE and protein was used

without further purification.

ELISA experiment 39,40

Non-competitive ELISA: The wells of a microtiterplate were coated with the 5HB

overnight at 4 ºC. After washing wells out with TBS-T and blocking with 5% milk in

TBS-T for 1 - 2 hrs at 4 ºC, serially diluted Trx-C49 was added into each well followed

by 2 hrs of the incubation at room temperature with agitation. Unbound Trx-C49 was

then washed away and a Trx-C49 specific anti-Stag antibody conjugated to alkaline

phosphatase (AP) was added and the plate was incubated for 1 hr at room temperature.

After the addition of the AP substrate, p-nitrophenyl phosphate (pNPP), absorbance at

405 nm was measured.

Competitive ELISA: The wells of a microtiterplate were coated with the 5HB followed

by the incubation with sample solution containing 4 nM of Trx-C49 and increasing

concentrations of Fl-C35. Unbound Trx-C49 was then rinsed off and a Trx-C49 specific

anti-Stag antibody conjugated to AP was added. Subsequent steps are the same as

described above.



51

Circular Dichroism spectroscopy

CD spectra were obtained with a Jasco J-815 spectrophotometer (JASCO, Easton,

MD, USA). Sample was prepared in 20 mM PBS, pH 7.4 in concentration of 35 µM.

Data were recorded from 195 to 260 nm with a scanning speed of 20 nm/min and a

bandwidth at 1.0 nm in a 0.1 cm path-length quartz cell. Each CD spectrum was an

average of 3 measurements and corrected for buffer blank obtained under identical

conditions. The resulting data was converted to per-residue molar ellipticity units, [!]

(deg cm2 dmol"1 residue-1), and the secondary structure content was analyzed with the

Dichroweb software package. The thermal stability of 5HB was monitored by

measuring its molar ellipticity between 0 °C and 85 °C at 222 nm. 2.5 µM of 5HB was

used for this study. The rate of temperature change was 2.5 °C/min with a scanning

speed of 50 nm/min and a bandwidth of 1.0 nm.

Peptide synthesis

Peptide synthesis reagents were purchased from Applied Biosystems (Foster city, CA,

USA) or Sigma-Aldrich (Milwaukee, WI, USA). Resins and Fmoc-protected amino

acids were purchased from NovaBioChem (San Diego, CA, USA) or Anaspec (San

Jose, CA, USA). Solvents for HPLC were purchased from Fisher Scientific

(Pittsburgh, PA, USA). All chemicals were used without additional purification.

Fluorescently-labeled peptide (Fl-C35) and truncated peptides (C30 and C35) of 95%

purity were commercially obtained (EZBiolab, Carmel, IN, USA and Bio Basic,

Markham, ON, Canada) and used without further purification. The remaining

truncated peptides (C20, C17 and N15) were synthesized in the laboratory using standard



52

Fmoc chemistry on solid support (preloaded Wang resin, Novabiochem, San Diego,

CA, USA) with an ABI 433A automated peptide synthesizer (Applied Biosystems,

Foster city, CA, USA). After synthesis, the peptides were cleaved from the resin and

deprotected in trifluoroacetic acid (TFA)/water/triisopropylsilane (TIPS)/thionisole

(90:5:2.5:2.5 v/v) for 1.5 hr at room temperature. Peptides were purified by

preparation RP-HPLC on a C18 column using a linear gradient of 5-99% solvent B in

solvent A over 60 min (solvent A is 0.1% (v/v) TFA in water and solvent B is 0.1%

(v/v) TFA in acetonitrile). Final purities of synthetic peptides were confirmed to be >

95% by analytical RP-HPLC and the molecular weight of the purified product was

confirmed by electrospray mass spectrometry (ESI) at the Stanford University Mass

Spectrometry (SUMS) facility.

FP measurements

FP measurements were performed using a Synergy4 (Biotek, Winooski, VT, USA)

plate reader with a tungsten lamp as a light source with an excitation wavelength of

485 nm and an emission wavelength of 530 nm. Fluorescently labeled 35 aa peptide,

Fl-C35, was chosen as a tracer due to its inhibitory potency (EC50 of 0.051 µM).6

Lyophilized Fl-C35 was dissolved in 20 mM PBS (at pH 7.4) and subsequent dilutions

were carried out in FP buffer (20 mM PBS at pH 7.4, 500 mM NaCl, 0.01% (v/v)

Tween-20, and 0.05 mg/ml bovine gamma globulin [BGG]). Specific control groups

included free Fl-C35 (Fl-C35 in the absence of 5HB, negative control), bound Fl-C35

(Fl-C35 in the presence of 5HB, positive control), and FP buffer for every

measurement, allowing accurate estimation of specific polarization.



53

Saturation Binding FP assays: The saturation binding experiments of Fl-C35 to 5HB

were performed under the following condition: each well in a black 96-well plate

(Corning Inc. Lowell, MA, USA) contained a final concentration of 5 nM of Fl-C35

(tracer) and increasing concentrations ranging from 0 to 500 nM of 5HB in a final

volume of 185 µL in FP buffer. The polarization in millipolarization units (mP) was

measured after 1 hr incubation at room temperature. Data obtained were analyzed

using GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA) to calculate a binding

dissociation constant (Kd) by fitting the experimental data using a one-site specific

binding model. Experiments were performed in duplicate.

Competitive FP binding assays: Each well in a black 96-well plate (Corning Inc.

Lowell, MA, USA) contained 20 nM of 5HB and increasing concentrations (0.001 to

200 µM) of each truncated peptide in FP buffer in a final volume of 185 µL. After 1hr

incubation at room temperature, Fl-C35 was added to 5 nM followed by 30 min

incubation at room temperature. The FP response was measured in duplicate with

controls including free Fl-C35, bound Fl-C35, and FP buffer. All experimental data

were plotted using GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). The

percentage of inhibition (% Inhibition) was calculated using the following equation:

% Inhibition = 100)[(mP-mPf)/(mPb-mPf)]

where mPf is the millipolarization of the free Fl-C35 control, mPb is the

millipolarization of the bound Fl-C35 control and mP is the millipolarization of the

bound inhibitor to the 5HB.



54

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47. Zhang, J.H., Chung, T.D. & Oldenburg, K.R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J

Biomol Screen 4, 67-73 (1999).

48. Zhao, X., Singh, M., Malashkevich, V.N. & Kim, P.S. Structural characterization of the human respiratory syncytial virus fusion protein core. Proc Natl Acad Sci U S A 97, 14172-14177 (2000).



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49. Kirschner, A.N., Lowrey, A.S., Longnecker, R. & Jardetzky, T.S. Binding-site interactions between Epstein-Barr virus fusion proteins gp42 and gH/gL reveal a peptide that inhibits both epithelial and B-cell membrane fusion. J Virol 81, 9216-9229 (2007).

50. Liu, F., Marquardt, G., Kirschner, A.N., Longnecker, R. & Jardetzky, T.S. Mapping the N-terminal Residues of EBV gp42 that Bind gH/gL Using Fluorescence Polarization and Cell-based Fusion Assays. J Virol (2010).

51. Bonfanti, J.F. & Roymans, D. Prospects for the development of fusion inhibitors to treat human respiratory syncytial virus infection. Curr Opin Drug

Discov Devel 12, 479-487 (2009).

52. Assay Guidance Manual Version 5.0, Eli Lilly and Company and NIH Chemical Genomics Center. Available online at: http://www.ncgc.nih.gov/ guidance/manual_toc.html (Dec.8, 2009)).

53. Roymans, D., De Bondt, H.L., Arnoult, E., Geluykens, P., Gevers, T., Van Ginderen, M., Verheyen, N., Kim, H., Willebrords, R., Bonfanti, J.F., Bruinzeel, W., Cummings, M.D., van Vlijmen, H. & Andries, K. Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein. Proc Natl

Acad Sci U S A 107, 308-313 (2010).

54. Bonfanti, J.F., Meyer, C., Doublet, F., Fortin, J., Muller, P., Queguiner, L., Gevers, T., Janssens, P., Szel, H., Willebrords, R., Timmerman, P., Wuyts, K., van Remoortere, P., Janssens, F., Wigerinck, P. & Andries, K. Selection of a respiratory syncytial virus fusion inhibitor clinical candidate. 2. Discovery of a morpholinopropylaminobenzimidazole derivative (TMC353121). J Med Chem 51, 875-896 (2008).

55. Cianci, C., Yu, K.L., Combrink, K., Sin, N., Pearce, B., Wang, A., Civiello, R., Voss, S., Luo, G., Kadow, K., Genovesi, E.V., Venables, B., Gulgeze, H., Trehan, A., James, J., Lamb, L., Medina, I., Roach, J., Yang, Z., Zadjura, L., Colonno, R., Clark, J., Meanwell, N. & Krystal, M. Orally active fusion inhibitor of respiratory syncytial virus. Antimicrob Agents Chemother 48, 413-422 (2004).



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56. Cianci, C., Meanwell, N. & Krystal, M. Antiviral activity and molecular mechanism of an orally active respiratory syncytial virus fusion inhibitor. J

Antimicrob Chemother 55, 289-292 (2005).

57. Razinkov, V., Huntley, C., Ellestad, G. & Krishnamurthy, G. RSV entry inhibitors block F-protein mediated fusion with model membranes. Antiviral

Res 55, 189-200 (2002).

58. McKimm-Breschkin, J. VP-14637 ViroPharma. Curr Opin Investig Drugs 1, 425-427 (2000).

59. Douglas, J.L., Panis, M.L., Ho, E., Lin, K.Y., Krawczyk, S.H., Grant, D.M., Cai, R., Swaminathan, S., Chen, X. & Cihlar, T. Small molecules VP-14637 and JNJ-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms. Antimicrob Agents Chemother 49, 2460-2466 (2005).

60. Bond, S., Draffan, A. & Lambert, J. Discoverty of a new class of polycyclic RSV inhibitors. Antivir Res 74, 9 (2007).

61. Sorensen, H.P. & Mortensen, K.K. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4, 1 (2005).

62. LaVallie, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F. & McCoy, J.M. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N Y) 11, 187-193 (1993).

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Chapter Three

NMEGylation: A novel modification to enhance the bioavailability of

therapeutic peptides!

Limitations in using synthetic peptides as therapeutics

With the monumental advances in biotechnology over the past few decades,

peptides and proteins have become key players in the drug market as therapeutic

candidates with undeniably high specificity and low toxicity compared to conventional

synthetic small molecule drugs, resulting in more than 60 biotherapeutics with sales

reaching US $40 billion.1,2 However, several unfavorable biophysical properties still

remain as challenges for clinical uses and manufacturing of peptide/protein-based

medicines. One of the major obstacles for the peptide-based therapeutics is that they

are extremely susceptible to proteolytic degradation, resulting in rapid renal clearance

with short in vivo circulation half-lives (2 – 5 min). Efforts have been made to increase

the half-lives of biotherapeutics without sacrificing their efficacy, but these efforts

have yielded only a few notable successes.3-8

PEGylation and its pressing challenges

PEGylation, the conjugation of PEG (poly-ethylene glycol) chains to

druggable materials, has been intensively studied, clinically proven and is an

acceptable method for modifying peptide/protein-based medicines to increase their

! This chapter is adapted from the following publication:

Park, M. et al. (2011) “NMEGylation: A novel modification to enhance the bioavailability of

therapeutic peptides” Biopolymers (Peptide Science). Accepted

Chapter Three: NMEGylation as a potential method for enhancing the bioavailability of peptide-

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protease resistance. The decrease in proteolysis reduces renal clearance rates and

enhances therapeutic efficacy. PEGylation is known to improve physical and thermal

stability as well as solubility of biopharmaceuticals, which makes PEGylation suitable

for drug delivery and formulation.9-12 Since Adagen" (PEGylated adenosine

deaminase, Enzon) was introduced as the first FDA-approved PEGylated therapeutic

agent in 1990, several PEGylated protein-based drugs have reached the market,

including PEGasys" (PEGylated #-interferons for Hepatitis C treatment,

Hoffmann/Roche) and Cimzia" (PEGylated Anti-TNF Fab for rheumatoid arthritis

and Crohn’s disease, UBC) (Table 3-1).9,13

Table 3-1. Marketed PEGylated biotherapeutics9,13

Name Company Target diseases Target protein Year to

market

Adagen® Enzon Severe combined

immunodeficiency Adenosine deamidase 1990

Oncaspar" Enzon Acute lymphobalstic leukemia Asparaginase 1994

Doxil" Novartis Kaposi’s sarcoma and ovarian

cancer

PEGylated liposomal

doxorubicin 1995

PEGasys" Hoffmann

/Roche Hepatitis C IFNa-#2a 2002

Neulasta" Amgen

/Nektar Neutropenia G-CSFb 2002

Macugen" Pfizer Ocular vascular disease Anti-VEGFc aptamer 2004

Cimizia" UCB Rheumatoid arthritis and

Crohn’s disease Anti TNFd Fab 2008

aIFN = Interferon bG-CSF = Granulocyte colony-stimulating factor cVEGF = Vascular endothelial growth factor dTNF = Tumor necrosis factor


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PEGylation, however, still presents challenges such as the heterogeneity

(polydispersity) of the PEG polymers and as a result, the mixture of PEGylated

products has become an issue in clinically using PEGylated drugs.14 To avoid variable

extent in PEGylation, PEGylation requires a complicated and multi-step

manufacturing process, which leads to higher costs.15 In addition to many different

approaches to improve specificity and homogeneity in PEGylation,16,17 efforts to

develop alternatives to PEG are also being pursued, resulting in currently limited but

promising outcomes.18-20

A novel peptide/protein modification: NMEGylation

N-methoxyethylglycine (NMEG) is a hydrophilic peptoid monomer and has a

similar chemical moiety (ethylene oxide unit) to that of PEG (Fig. 3-1). Peptoids are

peptidomimetics based on a peptide backbone that can resist proteolytic degradation

due to the peptoid side chains being attached to the backbone nitrogen instead of the

!-carbon (Fig. 3-2).21 Over the past few years peptoids have been widely used in many

biological research areas including gene delivery,22 drug delivery,23 and ligand design

with improved binding affinity.24-26 Any primary amine can be easily incorporated into

the peptoid backbone using a straightforward submonomer approach, which has

(A) (B)

HOO

HN

OCH3

O

n

n

Figure 3-1. Structure of (A) N-methoxyethylglycine (NMEG) and (B) poly-ethylene glycol (PEG)


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enabled the use of a diverse set of side chains in the peptoid research (Fig. 3-2).27

Previously the Barron group has reported that oligoNMEG (n = 10, 20 and 30)

can serve as (1) antifouling agents, which prevent non-specific protein binding to

surfaces28 and (2) friction-generating moieties for solution-based DNA separations.29

Even though NMEGylation, a covalent attachment of oligoNMEG, should provide

protease resistance to peptide/protein drugs,30,31 this possibility has not yet been

investigated. Thus, we explored NMEG as a promising PEG-like material in this

study. We previously identified a C20 peptide as a potential antiviral agent using a

protein-based screening assay as a part of an effort to identify potential viral entry

inhibitors targeting human respiratory syncytial virus (hRSV) infection (see Chapter

2).32 The C20 peptide showed a low micromolar binding affinity to the 5HB; however,

the solubility and stability of this short peptide are not ideal. Therefore, using the C20

peptide as a parent peptide, a series of NMEGylated C20 analogs were prepared with

different numbers of NMEG monomers and their biophysical properties and biological

activities were examined. We believe these results may open a new opportunity to use

peptoids as a modification method for biotherapeutics with broad applications in

biology and medicine.


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Figure 3-2. Synthetic approaches to prepare peptoids, peptides and peptomers (R = peptoid side chain, R’ = peptide side chain, Prt = protecting group, DIC = N,N!-diisopropyl carbodiimide, DMF = dimethylformamide, NMP = methylpyrrolidone, SPPS = Solid phase peptide synthesis)


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Synthesis, purification and characterization of NMEGylated C20 peptides

The C20 peptide was previously identified from peptide truncation studies

showing a low micromolar binding affinity to a 5-Helix Bundle (5HB), a genetically

engineered protein construct derived from hRSV fusion protein F;32 however, because

of its relatively short length and unstructured conformation,33-35 the C20 peptide has a

high susceptibility to proteases and poor solubility in aqueous solution. Although

PEGylation may improve the desirable biophysical features of the C20 peptide,

PEGylation was not pursued because the large increase in molecular weight of the

PEGylated C20 products and potential steric shielding might impede the binding of C20

to the 5HB. Thus we decided to explore NMEG as a potential alternative to PEG,

because NMEG is hydrophilic like PEG but monodisperse compared with many other

commercially available PEG derivatives. Importantly, because peptoids (e.g., NMEG)

cannot be recognized by proteases, we anticipated that the resulting peptoid-peptide

hybrids (peptomers) formed using NMEGylation would be less susceptible to

proteolytic degradation. As shown in Table 3-2, a series of NMEGylated C20 peptides

were prepared by attaching NMEG oligomers (n = 1 – 10) at either the N- or C-

terminus of C20. The resulting NMEGylated peptides were extremely soluble in

aqueous buffer (up to > 10 mg/mL) compared to C20 (< 2 mg/mL). The relative

hydrophilicity of NMEGylated C20 analogs based on percent acetonitrile at elution was

evaluated by RP-HPLC (Table 3-2).36,37 The decrease in percent acetonitrile of at

elution of NMEGylated analogs demonstrates that NMEGylation dramatically

enhances the hydrophilicity of the C20 peptide, notably, even with very short

NMEGylation (n = 1)


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Table 3-2. NMEGylated C20 peptomer sequences tested in this study with molecular weight (MW), purity, solubility, hydrophilicity and their IC50 values


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Biological activity of NMEGylated C20 peptides

Despite greatly increased hydrophilicity and solubility, NMEGylation of C20

with more NMEG monomers (n = 3, 5, and 10) was accompanied by significant loss

of binding affinity to the target protein, 5HB, as evaluated using our previously

reported protein-based fluorescence polarization (FP) assay (Table 3-2).32 Since the

binding of C20 to the 5HB involves a large interface, steric hindrance caused by the

attached NMEG oligomers might be occurring, suggesting the need for optimizing the

number of NMEG oligomers. Therefore, NMEGylated peptides with only one NMEG

at either or both termini of the C20 peptide were prepared, and their biophysical

properties and biological activities against the 5HB construct were evaluated.

Although addition of even a single NMEG to C20 greatly improves solubility and

hydrophilicity, binding affinity is still significantly diminished (Fig. 3-3, Table 3-2).

Figure 3-3. Resulting data from competitive FP assays Binding of NMEGylated C20 peptomers to the 5HB was determined using FP. % Inhibition represents the displacement capability of NMEGylated peptomers over a tracer (Fl-C35) to the target protein (5HB) and is presented as a function of the concentration of NMEGylated C20 peptides.


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To recover the biological activity of NMEGylated C20, we decided to use an

NMEG bound through a flexible linker to allow the C20 enough space to properly form

#-helical conformation upon binding to the 5HB. On the other hand, a linker that is

too flexible would lose too much entropy upon binding and consequently deleteriously

affect binding affinity. Glycine was chosen as the linker because we anticipated that

glycine would be flexible but would not disrupt the peptide backbone structure. To

determine the number of glycines that maximizes C20 binding to the 5HB, N-

terminally NMEGylated C20 (n = 1) with different numbers of glycine residues (n = 0,

1, 2, and 3) were synthesized and the binding affinity of each peptomer was examined

(Fig. 3-4).

NMEG-glycine-C20 (NMEGGC20) showed significantly tighter binding affinity

to the 5HB than the rest of the NMEGylated C20 analogs with greatly improved

solubility and hydrophilicity (Table 3-2, Fig. 3-4A). Because glycine is known to be a

(A) (B)

Figure 3-4. Optimization of the length of the glycine linker (A) Binding NMEGylated C20 peptomers with different numbers of glycines to the 5HB were determined using

competitive FP assay. (B) Comparison of 5HB•Fl-C35 bound in the presence of peptomers

(100 µM) with a different number of glycines residues as linkers. The binding affinity of the

parent C20 is shown in blue for ease of comparison.


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helix breaker,38,39 it is possible that longer glycine linkers might impede the binding

affinities of peptomers to the 5HB by increasing the flexibility of the peptide backbone

structure (Fig. 3-4B). The benefits of NMEGylation of potential biotherapeutics

should increase when the NMEG attachment points directly away from the likely

binding interface. Since the C20 peptide likely forms an #-helix upon binding to the

5HB,40 a direct attachment of peptoid residues may disrupt the #-helix formation due

to the lack of structural rigidity.41 The secondary structural analysis of the

NMEGylated peptomers will be discussed later in this chapter.

Serum stability of NMEGylated C20 peptides

As discussed earlier, PEGylation is clinically proven to extend the half-life of

biotherapeutics, thereby improving their efficacy. We therefore examined whether

NMEGylation can provide a similar beneficial effect, focusing on singly-NMEGylated

peptomers with greatly improved aqueous solubility. NMEGylated C20 peptides were

incubated with human serum, which contains many proteases (e.g., trypsin and

elastase), at room temperature and then analyzed the mixture by RP-HPLC to

determine the amount of remaining intact peptomers. Our resulting data (Fig. 3-5)

indicate that NMEGylation indeed enhanced the serum stability of NMEGylated

peptides compared to the parent peptide, C20. Interestingly, the protease stability of

NMEGGC20 was the most greatly improved, suggesting that despite a slight loss of

binding affinities, NMEGGC20 could present comparable therapeutic efficacy to the

C20 peptide due to a prolonged half-life.


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Secondary structure analysis of NMEGylated C20 peptides in different solvent

systems

As mentioned earlier in this chapter, the C20 peptomers with longer NMEG

chains showed diminished binding to the 5HB, although they possessed greatly

enhanced solubility and hydrophilicity compared to the parent peptide, C20. To better

understand how NMEGylation influences the secondary structures of peptomers, CD

spectra of NMEGylated peptomers were monitored in different solvent systems such

as aqueous solution (e.g., Tris) or mixtures of aqueous and organic solvents (e.g., 50%

acetonitrile in Tris). Per residue molar ellipticity ($, deg%cm2%dmol-1) of NMEGylated

C20 analogs (100 µM) was measured at 222 nm, and the results of these studies

Figure 3-5. Peptide stability in presence of serum NMEGylated peptides were incubated with human plasma at 37 °C and sampled at the indicated time points, followed by RP-HPLC analysis. The amount of remaining peptomer at each time point was quantified against an enzyme-stable internal organic standard (benzyl alcohol).


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suggest that the longer the NMEG chain, the more irregular the secondary structure

(Fig. 3-6). Attachment of one NMEG does not perturb the secondary structure of the

C20 peptide, whereas longer oligoNMEG (n > 3) have a significantly greater impact on

the C20 structure. These observations provided useful information to correlate the

probable structures of these peptomers with their binding affinities to the 5HB.

We also investigated the solvent polarity dependency of NMEGylated C20 in

different solvent systems, focusing on C20NMEG3 as intermediate in length of

NMEGylation and secondary structure. Due to poor aqueous solubility, stock solution

of the C20 peptide were prepared in organic solvent (e.g., acetonitrile) and then diluted

in an aqueous solution (e.g. Tris) to obtain the desired concentrations. Since

acetonitrile is less polar then water, we varied the concentration of acetonitrile in Tris

buffer to alter the polarity of the solvent mixture. The resulting CD spectra of

C20NMEG3 in Fig. 3-7 indicate that as the concentration of acetonitrile decreased, the

Figure 3-6. Comparison of helical propensity of NMEGylated C20 peptomers with C20

peptide at 222 nm 100 µM of each compound was prepared in 10 mM TBS containing 50

mM NaCl at pH 7.4 and CD spectra at 222 nm were obtained at room temperature.


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NMEGylated peptomer tends to show an increase in the intensity of the very strong

band at 200 nm band and a decreased intensity in the band at 222 nm, consistent with

possible loss of a regular secondary structure. Additionally, the spectra were shifted to

shorter wavelengths (blue shift) when the solvent polarity increased, suggesting the

destabilization of structured conformations.42

To examine the possibility of aggregation of the NMEGylated peptomers that

might lead to unexpected structure formation, the concentration dependence of the

secondary structure of C20NMEG3 was tested. However, neither concentration-

dependent structural changes nor signs of aggregation were observed (Fig. 3-8),

suggesting that the NMEGylated peptomers are present in monomeric forms and that

Figure 3-7. Solvent polarity dependency in CD spectra of 100 µM of C20NMEG3 The

polarity of the solvent mixture is increased from 0 to 75% acetonitrile, which are indicated in dotted black, solid gray, and black lines. Blue arrow indicates a blue shift of 200 nm-negative band and red arrow represents a decrease in the intensity of 222 nm-negative band.


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the secondary structure of NMEGylated C20 peptomers might be governed primarily

by the solvent polarity rather than by undesirable aggregation.

(A) (B)

(C)

Figure 3-8. CD spectra of different concentrations of C20Nmeg3 ranging from 10 to

200 µM in various solvent systems (A) 0%, (B) 50%, and 75% acetonitrile in Tris

buffer


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In this study, we demonstrate that NMEGylation enhances multiple

therapeutically desired properties of peptides including the solubility, hydrophilicity

and serum stability. Our data also suggest that modifying the molecular weight of the

target peptide, C20, by less than 5% with NMEGylation is sufficient to achieve

favorable biophysical properties. Furthermore, optimizing the length of the NMEG

and glycine linkers enabled the NMEGylated peptomer to greatly recover the binding

affinity to its biological target. While PEGylation is still a useful technique for large

proteins, it requires post-synthetic conjugation and multiple purification steps to obtain

the desired PEGylated products.13 In contrast, NMEGylation can be used to modify

short therapeutic peptides as well as proteins, quickly yielding highly monodisperse

products at lower costs. Since NMEGylation is intrinsically compatible with solid

phase peptide/peptoid synthesis and offers both low-cost, short synthesis times, and

site-specific incorporation as part of the synthesis protocols, NMEGylated

peptide/peptoid libraries could be designed at an early stage of molecular optimization

by varying both the number and position of NMEG monomers and linker units.

Therefore, NMEGylation may prove a new, broadly useful method of peptoids for

future modification of therapeutically attractive peptides and proteins.


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Methods and Materials

General materials

Reagents for peptide and peptomer synthesis were purchased from Applied

Biosystems (Foster city, CA, USA) or Sigma-Aldrich (Milwaukee, WI, USA). Resins

and Fmoc-protected amino acids were purchased from NovaBioChem (San Diego,

CA, USA) or Anaspec (San Jose, CA, USA). Solvents for analytical and preparative

RP-HPLC were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other

chemicals were purchased from Sigma-Aldrich (Milwaukee, WI, USA) and used

without additional purification.

NMEGylated peptomers and C20 peptide synthesis

The C20 and its NMEGylated analogs were synthesized in the laboratory using

standard Fmoc chemistry and a submonomer peptoid synthesis27 (Fig. 3-2) with an

automated ABI 433A peptide synthesizer (Applied Biosystems, Foster city, CA,

USA). After synthesis, the peptides were cleaved off the resin and deprotected in

trifluoroacetic acid (TFA)/water/triisopropylsilane/thionisole (90:5:2.5:2.5 v/v) for 1.5

hr at room temperature with agitation. Peptide and peptomers were purified by

preparative RP-HPLC on a C18 column using a linear gradient of 5-99% solvent B in


(v/v) TFA in acetonitrile). Final purities of the synthetic peptides were confirmed to be

> 95% by analytical RP-HPLC, and the molecular weight of the purified product was

confirmed by electrospray mass spectrometry (ESI) at the Stanford University Mass

Spectrometry facility.


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Selected structure of NMEGylated peptomers (A; C20, B; NmgGC20, C;

NMEG3C20, and D; C20NMEG (C20 peptide in black, glycine spacer in blue and NMEG in red)


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Selected HPLC traces of NMEGylated peptomers (A; C20, B; NmgGC20, C;

NMEG3C20, and D; C20NMEG3)


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Percent acetonitrile measurement

The relative hydrophilicity (percent acetonitrile at elution) of NMEGylated peptides

was compared by analytical RP-HPLC, previously reported as a reliable method for

determining the contribution of the hydrophilicity of amino acids to the retention time

of peptides.36,37 The purified C20 and its NMEGylated analogs were analyzed using

RP-HPLC on a Phenomenex (Torrance, CA, USA) C18 column (250 mm x 2.00 mm)

with 5 µm resin size using a linear gradient of 5-95% solvent B over 30 or 60 min

(solvent A is water with 0.1% (v/v) TFA and solvent B is acetonitrile with 0.1% (v/v)

TFA).

Serum stability assay43

Peptides (~ 0.4 mg) were dissolved in 20 mM PBS at pH 7.6 (400 µL containing 0.1%

v/v benzyl alcohol as internal standard). Equal volumes of 50% human serum plasma

(Sigma-Aldrich, Milwaukee, WI, USA) and peptides solutions were combined and

incubated at 37°C. Samples were taken in triplicate (3 " 50 µL) at 0, 30, and 240 min

and placed on ice. Each sample (including the t = 0 time point) was prepared for

HPLC analysis in the following way. First, 20 µL of 0.5 M lysine monohydrochloride

was added, followed by 65 µL of acetonitrile. The samples were then cooled on ice for

10 min and subsequently centrifuged for 10 min at 3000 rpm. A 50 µL aliquot of the

resulting samples was taken from the supernatant, diluted with 100 µL of water and

analyzed by analytical RP-HPLC. The ratio of the disappearing peptomer peak area

relative to the benzyl alcohol peak area was calculated for each time point and


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normalized against the t = 0 time point (representing 100% peptide remaining) as a

percentage of the peptide remaining.

Competitive fluorescence polarization (FP) binding assay

Competitive FP binding assays of NMEGylated C20 peptomers were carried out as

previously descibed.32 Briefly, using a 5HB protein construct and a fluorescently

labeled 35 amino acid-long peptide (Fl-C35: YDPLVFPSDEFDASISQVNEKINQSL

AFIRKSDEL) as a target and a tracer respectively, the binding affinities of

NMEGylated peptomers were individually evaluated. Both the C20 and C35 peptide

sequences are derived from HRB domain of hRSV F protein and mimic the 6-helix

bundle formation of hRSV F protein by occupying the binding site on the 5HB. The

binding of Fl-C35 peptide to the 5HB in absence of peptomers provides 100% 5HB%Fl-

C35 bound as a positive control. Each well in a black 96-well plate (Corning Inc.

Lowell, MA, USA) contained 20 nM of the 5HB and increasing concentrations of each

NMEGylated peptomer in FP buffer (20 mM PBS at pH 7.4, 500 mM NaCl, 0.01%

(v/v) Tween-20, and 0.05 mg/ml bovine gamma globulin) in a final volume of 185 µL

with 1 hr incubation at room temperature. Fl-C35 was then added to a final

concentration of 5 nM followed by 30 min incubation at room temperature. The FP

responses were monitored using a Synergy4 plate reader (Biotek, Winooski, VT,

USA) with an excitation wavelength of 485 nm and an emission wavelength of 530

nm. All experimental data were obtained in duplicate and the percentage of inhibition

(% Inhibition) was calculated using the following equation: % Inhibition = 100&[(mP-


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mPf)/(mPb-mPf)], where unbound Fl-C35 (mPf), bound Fl-C35 (mPb) and the bound

inhibitor to the 5HB (mP) are used accordingly.

Secondary structural analysis by CD spectroscopy


MD, USA). Sample was prepared in 10 mM Tris containing 50 mM NaCl (pH 7.4) in

concentrations ranging from 10 – 200 µM. In some cases, 50 – 75% of acetonitrile

was used to test solvent effect on the secondary structures of NMEGylated peptomers.

Data were recorded from 195 to 260 nm with a step size of 0.2 nm, at a rate of 100

nm/min, a bandwidth of 1.0 nm with a scanning speed of 20 nm/min in a 0.1 cm path-

length quartz cell. The response time was 0.5 s. Each CD spectrum was an average of

3 measurements and the baseline was corrected by subtracting the spectrum of a buffer

blank obtained under identical conditions. The resulting data was converted to per-

residue molar ellipticity units, [#] (deg cm2 dmol-1 residue-1).


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13. Jevsevar, S., Kunstelj, M. & Porekar, V.G. PEGylation of therapeutic proteins. Biotechnol J 5, 113-128 (2010).

14. Fee, C.J. & Van Alstine, J.M. PEG-Proteins: Reaction engineering and separation issues. Chem Eng Sci 61, 924-939 (2006).

15. Grace, M.J., Lee, S., Bradshaw, S., Chapman, J., Spond, J., Cox, S., Delorenzo, M., Brassard, D., Wylie, D., Cannon-Carlson, S., Cullen, C., Indelicato, S., Voloch, M. & Bordens, R. Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway. J Biol Chem 280, 6327-6336 (2005).

16. Shaunak, S., Godwin, A., Choi, J.W., Balan, S., Pedone, E., Vijayarangam, D., Heidelberger, S., Teo, I., Zloh, M. & Brocchini, S. Site-specific PEGylation of native disulfide bonds in therapeutic proteins. Nat Chem Biol 2, 312-313 (2006).

17. Brocchini, S., Godwin, A., Balan, S., Choi, J.W., Zloh, M. & Shaunak, S. Disulfide bridge based PEGylation of proteins. Adv Drug Deliv Rev 60, 3-12 (2008).

18. Perrino, C., Lee, S., Choi, S.W., Maruyama, A. & Spencer, N.D. A biomimetic alternative to poly(ethylene glycol) as an antifouling coating: resistance to nonspecific protein adsorption of poly(L-lysine)-graft-dextran. Langmuir 24, 8850-8856 (2008).

19. Han, J.O., Joo, M.K., Jang, J.H., Park, M.H. & Jeong, B. PVPylated poly(alanine) as a new thermogelling polymer. Macromolecules 42, 6710-6715 (2009).

20. Schellenberger, V., Wang, C.W., Geething, N.C., Spink, B.J., Campbell, A., To, W., Scholle, M.D., Yin, Y., Yao, Y., Bogin, O., Cleland, J.L., Silverman, J. & Stemmer, W.P. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol 27, 1186-1190 (2009).


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21. Patch, J.A., ;Kirshenbaum, K.; Seurynck, S.L.;Zuckermann, R.N.;Barron, A.E. In Pseudopeptides in Drug Development, (Wiley-VCH, Weinheim, Germany, 2004).

22. Murphy, J.E., Uno, T., Hamer, J.D., Cohen, F.E., Dwarki, V. & Zuckermann, R.N. A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery. Proc Natl Acad Sci U S A 95, 1517-1522 (1998).

23. Wender, P.A., Mitchell, D.J., Pattabiraman, K., Pelkey, E.T., Steinman, L. & Rothbard, J.B. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci

U S A 97, 13003-13008 (2000).

24. Nguyen, J.T., Porter, M., Amoui, M., Miller, W.T., Zuckermann, R.N. & Lim, W.A. Improving SH3 domain ligand selectivity using a non-natural scaffold. Chem Biol 7, 463-473 (2000).

25. Nguyen, J.T., Turck, C.W., Cohen, F.E., Zuckermann, R.N. & Lim, W.A. Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors. Science 282, 2088-2092 (1998).

26. Zimmermann, J., Kuhne, R., Volkmer-Engert, R., Jarchau, T., Walter, U., Oschkinat, H. & Ball, L.J. Design of N-substituted peptomer ligands for EVH1 domains. J Biol Chem 278, 36810-36818 (2003).

27. Zuckermann, R.N., Kerr, J.M., Kent, S.B.H. & Moos, W.H. Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis. J Am Chem Soc 114, 10646-10647 (1992).

28. Statz, A.R., Meagher, R.J., Barron, A.E. & Messersmith, P.B. New peptidomimetic polymers for antifouling surfaces. J Am Chem Soc 127, 7972-7973 (2005).

29. Haynes, R.D., Meagher, R.J., Won, J.I., Bogdan, F.M. & Barron, A.E. Comblike, monodisperse polypeptoid drag-tags for DNA separations by end-labeled free-solution electrophoresis (ELFSE). Bioconjug Chem 16, 929-938 (2005).


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30. Miller, S.M., Simon, R.J., Ng, S., Zuckermann, R.N., Kerr, J.M. & Moos, W.H. Comparison of the proteolytic susceptibilities of homologous L-amino acid, D-amino acid, and N-substituted glycine peptide and peptoid oligomers. Drug Dev. Res. 35, 20-32 (1995).

31. Miller, S.M., Simon, R.J., Ng, S., Zuckermann, R.N., Kerr, J.M. & Moos, W.H. Proteolytic studies of homologous peptide and N-substituted glycine peptoid oligomers. Bioorg Med Chem Lett 4, 2657-2662 (1994).

32. Park, M., Matsuura, H., Lamb, R.A., Barron, A.E. & Jardetzky, T.S. A fluorescence polarization assay using an engineered hRSV F protein as a direct screening platform Anal Biochem 409, 195-201 (2011).

33. Harrison, R.S., Shepherd, N.E., Hoang, H.N., Ruiz-Gomez, G., Hill, T.A., Driver, R.W., Desai, V.S., Young, P.R., Abbenante, G. & Fairlie, D.P. Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency. Proc Natl Acad Sci U S A 107, 11686-11691 (2010).


35. Matthews, J.M., Young, T.F., Tucker, S.P. & Mackay, J.P. The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil. J Virol 74, 5911-5920 (2000).

36. Parker, J.M., Guo, D. & Hodges, R.S. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites. Biochemistry 25, 5425-5432 (1986).

37. Sereda, T.J., Mant, C.T., Sonnichsen, F.D. & Hodges, R.S. Reversed-phase chromatography of synthetic amphipathic alpha-helical peptides as a model for ligand/receptor interactions. Effect of changing hydrophobic environment on the relative hydrophilicity/hydrophobicity of amino acid side-chains. J

Chromatogr A 676, 139-153 (1994).


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38. Chakrabartty, A., Kortemme, T. & Baldwin, R.L. Helix propensities of the amino acids measured in alanine-based peptides without helix-stabilizing side-chain interactions. Protein Sci 3, 843-852 (1994).

39. Javadpour, M.M., Eilers, M., Groesbeek, M. & Smith, S.O. Helix packing in polytopic membrane proteins: role of glycine in transmembrane helix association. Biophys J 77, 1609-1618 (1999).

40. Zhao, X., Singh, M., Malashkevich, V.N. & Kim, P.S. Structural characterization of the human respiratory syncytial virus fusion protein core. Proc Natl Acad Sci U S A 97, 14172-14177 (2000).

41. Fowler, S.A. & Blackwell, H.E. Structure-function relationships in peptoids: recent advances toward deciphering the structural requirements for biological function. Org Biomol Chem 7, 1508-1524 (2009).

42. Otvos, L., Jr. Use of circular dichroism to determine secondary structure of neuropeptides. Methods Mol Biol 73, 153-161 (1997).

43. Scanlon, D., Harris, K.S., Coley, A.M., Karas, J.A., Casey, J.L., Hughes, A.B. & Foley, M. Comprehensive N-Methyl Scanning of a Potent Peptide Inhibitor of Malaria Invasion into Erythrocytes Leads to Pharmacokinetic Optimization of the Molecule Int. J. Pept. Res. Ther. 14, 381-386 (2008).

87

Chapter Four

Knowledge-based approaches for designing peptoid-peptide hybrids

and their structurally constrained analogues

Background and motivation for this study

As discussed in Chapter 1, synthetic peptides have recovered their reputation

as therapeutics due to their undeniably advantageous features (e.g., high specificity)

for medical applications; however, manufacturing synthetic peptides is still hindered

by high production costs due to the laborious chemical synthesis. Additional

challenges for broader clinical use of therapeutic synthetic peptides are their poor

biophysical properties including rapid renal clearance, low cell-permeability, variable

solubility, high immunogenecity, and limited stability.1-3

We previously demonstrated

a fluorescence polarization assay using an engineered hRSV F protein, 5-Helix Bundle

(5HB), to screen potential hRSV fusion inhibitors and identified the C20 peptide,

which has a low micromolar binding affinity to the 5HB (see Chapter 2 for details).4

To enhance the biophysical properties of C20, we explored proteolytically resistant

peptoids (N-substituted glycines) for designing peptide-peptoid hybrid (peptomer)

hRSV inhibitors. As discussed in Chapter 1, peptoids are bioinspired polymers with

side chains attached to the peptide backbone nitrogen that possess many attractive

features such as altered conformations and chemical versatility.5 In Chapter 3, we

tested peptoid modification to peptides by decorating the “outside” of the C20 peptide

sequence (i.e., at N- or C-terminus).

Chapter Four: Knowledge-based approaches to design peptoid-peptide hybrids and their

structurally constrained analogues

88

In this chapter, we take this approach even further by incorporating peptoids

directly “within” the peptide sequences. Since there are no straightforward ways to

convert biologically active peptides into peptoids or peptomers with retention of

biological activity, often many variants have to be synthesized, tested, and

progressively optimized.6-9

This laborious and time-consuming approach has

unsurprisingly led to relatively few successes in transforming therapeutically

interesting peptides into peptoids.10-15

Thus, in addition to improving the biological

properties of the C20 peptide, we are at least equally interested in developing a

broadly-applicable approach for converting biologically active peptides into peptoids

and peptomers.

A combined approach of alanine, proline, and sarcosine scans

When substituting individual residues in peptides with regular amino acids, an

“alanine scan” is used to elucidate the relative importance of each side chain;16,17

a

series of analogues is prepared where each amino acid is replaced in turn by alanine,

which has a minimal side chain, and the impact on the activity of the peptide is

assessed. In replacing peptide residues with peptidomimetics, however, the peptide

backbone itself is typically altered, and thus more sophisticated approaches need to be

used.

Specifically, the substitution of peptoid residues into peptides must address

three criteria: first, the importance of the original amino acid and its side chain, as can

be elucidated with an alanine scan; second, the importance of the amide hydrogen,

which is missing in peptoids, and could be investigated using sarcosine (Fig. 4-1), the



89

peptoid equivalent of alanine; third, particularly if substituting peptoid residues into a

helix, the tolerance of the peptide !–helix for the peptoid polyproline type I (PPI)-like

helix structure needs to be assessed. Since peptoid helices form a PPI-like helix,

proline itself could quite reasonably serve as a probe of the suitability of the parent

helix to incorporate this structure. Whereas alanine scans are both routine and

ubiquitous, there are almost no literature reports of proline and sarcosine scans, and no

reports of combining these approaches.18-20

Therefore, we used the alanine, proline and sarcosine scans together on the C20

peptide, establishing a novel, comprehensive approach to identify peptoid-tolerable

residues in the C20 peptide. Substitution of each amino acid along the C20 peptide

sequence successively by alanine, proline and sarcosine was carried out, generating 60

peptides in total. After evaluating the binding affinity of these peptides to the 5HB

using our competitive FP assay as discussed below, we found that several substituted

peptides could maintain moderate 5HB-binding affinities. Subsequently, we

incorporated peptoid residues with identical side chains to the peptide amino acid

being replaced into the positions determined by the combined scans, creating

peptomeric anti-hRSV fusion inhibitors.

HN

CH3

O

N

O

H3C

N

O

Alanine Proline Sarcosine

Figure 4-1. Structural comparison of alanine, proline, and sarcosine



90

Although one could have theoretically utilized the peptoid residues directly

with the same side chains instead of sarcosine in a “peptoid scan”, we intentionally

used the sarcosine scan for two reasons. First, although peptoid synthesis is entirely

compatible with peptide synthesis, it is not as universally used; in contrast, sarcosine is

available as Fmoc-sarcosine and could thus be immediately used in solid phase

peptide synthesis protocols without modification, thereby enabling wider adoption of

this scanning technique. Second, although not pursued in this study, the data from this

combined approach could also serve as a starting point for a library screening

approach that would investigate peptoid side chains different from that of the original

amino acid being substituted and that would take advantage of the near limitless

chemical diversity of peptoids.

We believe that the combination of the alanine and sarcosine scans in

particular could become a universal approach for preparing peptoid and peptomer

therapeutics. Furthermore, our novel comprehensive approach could be applied to

other peptidomimetic systems with altered peptide backbones, such as by combining

alanine and "–alanine to generate "–alanine containing therapeutic peptides, or by

combining multiple such assays, e.g., alanine, sarcosine, and "–alanine scans to

determine the best peptidomimetic substitutions for any position in a therapeutic

peptide.



91

Alanine substitution study

To evaluate the importance of the side chains in the interaction of the C20

peptide with the 5HB, 20 alanine-substituted peptides were synthetically prepared

(Table 4-1) and the binding affinity of each peptide to the 5HB was examined at the

concentrations of 100 and 200 µM using the 5HB-based competitive FP assay. The

results were subsequently converted to % inhibition of Fl-C35 binding to the 5HB,

which is summarized in Table 4-1 and Fig. 4-2.

Table 4-1. Sequences of alanine-substituted peptides tested in this study and their inhibitory effect on binding of Fl-C35 to the 5HB at 200 µM

Peptide Sequences (amino to carboxy) MW (Da) % Inhibition

C20 ISQVNEKINQSLAFIRKSDE a d a d a d

2319.6 100

A1 ASQVNEKINQSLAFIRKSDE 2277.5 16

A2 IAQVNEKINQSLAFIRKSDE 2304.6 24

A3 ISAVNEKINQSLAFIRKSDE 2263.5 27

A4 ISQANEKINQSLAFIRKSDE 2291.8 40

A5 ISQVAEKINQSLAFIRKSDE 2277.6 26

A6 ISQVNAKINQSLAFIRKSDE 2261.6 89

A7 ISQVNEAINQSLAFIRKSDE 2262.5 100

A8 ISQVNEKANQSLAFIRKSDE 2277.5 14

A9 ISQVNEKIAQSLAFIRKSDE 2276.6 31

A10 ISQVNEKINASLAFIRKSDE 2262.6 11

A11 ISQVNEKINQALAFIRKSDE 2303.6 33

A12 ISQVNEKINQSAAFIRKSDE 2277.5 44

A13 ISQVNEKINQSLAFIRKSDE 2319.6 100

A14 ISQVNEKINQSLAAIRKSDE 2243.5 25

A15 ISQVNEKINQSLAFARKSDE 2277.5 21

A16 ISQVNEKINQSLAFIAKSDE 2234.5 54

A17 ISQVNEKINQSLAFIRASDE 2262.5 100

A18 ISQVNEKINQSLAFIRKADE 2303.6 37

A19 ISQVNEKINQSLAFIRKSAE 2275.6 32

A20 ISQVNEKINQSLAFIRKSDA 2261.6 29



92

Alanine substitutions at Glu6, Lys7, and Lys17 that point away from the

expected binding interface are the most well-tolerated, suggesting these peptide

residues could be candidates for peptoid substitution. Interestingly, we observed that

the further the alanine substitutions occur from the binding interface, the more well-

tolerated they are (Table 4-1). These observations are consistent with the extreme

stability of the helical bundle to denaturing (as discussed in Chapter 2) and suggest

that the hydrophobic residues involved in the direct interaction between the C20

peptide and the 5HB are critical for binding of the C20 peptide to the 5HB and should

not be altered.

Figure 4-2. % Inhibition of alanine-substituted peptides at 100 and 200 µM Each

peptide was competed with 5 nM Fl-C35 in the presence of 20 nM 5HB and its ability to displace Fl-C35 was monitored by FP. The FP response was converted to % inhibition.



93

Proline substitution study

As the only naturally occurring N-substituted amino acid, proline has been

recognized as natural candidate for peptoid substitution in various studies. For

example, prolines in WW and SH3 domain binding peptide ligands have been replaced

with various peptoid monomers leading to improved binding affinity.10,11

In addition,

proline substitution with peptoid monomers in proline-containing antimicrobial

peptides increased their antimicrobial activity21,22

as well as the binding affinity of

plant peptide hormones.14

Inspired by these exciting findings in using proline as a

prototypical peptoid, we substituted each amino acid in the C20 peptide with proline

one by one to identify peptoid-tolerant peptide residues. The resulting data shown in

Fig. 4-3 and Table 4-2 indicate that proline substitutions greatly detract from the

binding affinities of the C20 peptide analogues, yielding minimal % inhibition values

even at 200 µM.

Table 4-2. Sequences of proline-substituted peptides tested in this study and their

inhibitory effect on binding of Fl-C35 to the 5HB at 200 µM


P1 PSQVNEKINQSLAFIRKSDE 2303.5 26

P2 IPQVNEKINQSLAFIRKSDE 2329.6 23

P3 ISPVNEKINQSLAFIRKSDE 2288.5 14

P4 ISQPNEKINQSLAFIRKSDE 2317.5 12

P5 ISQVPEKINQSLAFIRKSDE 2302.6 34

P6 ISQVNPKINQSLAFIRKSDE 2287.6 26

P7 ISQVNEPINQSLAFIRKSDE 2288.5 -

P8 ISQVNEKPNQSLAFIRKSDE 2303.5 11

P9 ISQVNEKIPQSLAFIRKSDE 2302.6 33

P10 ISQVNEKINPSLAFIRKSDE 2288.5 9

P11 ISQVNEKINQPLAFIRKSDE 2329.6 9

P12 ISQVNEKINQSPAFIRKSDE 2303.5 22



94

P13 ISQVNEKINQSLPFIRKSDE 2345.6 10

P14 ISQVNEKINQSLAPIRKSDE 2269.5 13

P15 ISQVNEKINQSLAFPRKSDE 2303.5 4

P16 ISQVNEKINQSLAFIPKSDE 2260.5 4

P17 ISQVNEKINQSLAFIRPSDE 2288.5 6

P18 ISQVNEKINQSLAFIRKPDE 2329.6 0

P19 ISQVNEKINQSLAFIRKSPE 2301.6 84

P20 ISQVNEKINQSLAFIRKSDP 2287.6 31

The decreased binding affinities of proline-substituted peptides can be

explained by proline’s intrinsic structure. Proline is known to induce a kink in !-

helices, often leading to unusual conformations23

and significant flexibility in the !-

helix24-26

as well as the rapid cis/trans isomerization.27

Despite these reasons, the PPI-

like helical structure adopted by bulky !-chiral peptoid residues may have proved

entropically favorable, which is why the proline scan was pursued. It should be noted

Figure 4-3. % Inhibition of proline-substituted peptides at 100 and 200 µM Each




95

that the greater rigidity of the !-chiral peptoid residues should still be useful to

improve the binding affinity of non-helical peptides, and that a proline scan would be

more relevant in such systems. The findings from proline substitution studies on the

C20 peptide confirmed that maintaining !-helical conformation is crucial for the C20

peptide to bind to the 5HB and thus peptomers with !-chiral peptoid residues were not

synthesized. Notably, this is the first time that the hypothesis of proline as the

representative of structure-promoting peptoids has been tested in the context of

designing peptoid-based peptidomimetics.

Sarcosine substitution study

Sarcosine, also known as N-methylglycine, has already been used to improve

the pharmacokinetic properties of biologically intriguing peptides including metabolic

stability and permeability,28,29

and to test the tolerance to conformational changes in

short peptides.10,11,20

Moreover, as the simplest peptoid monomer, sarcosine has been

used to identify pharmacophores in peptoid ligands from combinatorial libraries of

peptoids, in a direct parallel of the alanine scan in peptides.30

We thus expected that

the sarcosine substitution study of the C20 peptide would reveal which peptide residues

can be replaced with peptoid monomers without severely impacting the binding

affinity to the 5HB. Twenty sarcosine-substituted peptides were commercially

obtained and their binding affinities to the 5HB were investigated using 5HB-based

competitive FP assays (Table 4-3 and Fig. 4-4).



96

Table 4-3. Sequences of sarcosine-substituted peptides tested in this study and their

inhibitory effect on binding of Fl-C35 to the 5HB at 200 µM


Sar1 SarSQVNEKINQSLAFIRKSDE 2277.5 15

Sar2 ISarQVNEKINQSLAFIRKSDE 2303.6 22

Sar3 ISSarVNEKINQSLAFIRKSDE 2262.6 11

Sar4 ISQSarNEKINQSLAFIRKSDE 2291.6 1

Sar5 ISQVSarEKINQSLAFIRKSDE 2276.6 23

Sar6 ISQVNSarKINQSLAFIRKSDE 2261.6 100

Sar7 ISQVNESarINQSLAFIRKSDE 2262.6 100

Sar8 ISQVNEKSarNQSLAFIRKSDE 2277.5 26

Sar9 ISQVNEKISarQSLAFIRKSDE 2276.6 13

Sar10 ISQVNEKINSarSLAFIRKSDE 2262.6 21

Sar11 ISQVNEKINQSarLAFIRKSDE 2303.6 13

Sar12 ISQVNEKINQSSarAFIRKSDE 2277.5 7

Sar13 ISQVNEKINQSLSarFIRKSDE 2319.6 4

Sar14 ISQVNEKINQSLASarIRKSDE 2243.5 4

Sar15 ISQVNEKINQSLAFSarRKSDE 2277.5 30

Sar16 ISQVNEKINQSLAFISarKSDE 2234.5 0

Sar17 ISQVNEKINQSLAFIRSarSDE 2262.5 84

Sar18 ISQVNEKINQSLAFIRKSarDE 2303.6 21

Sar19 ISQVNEKINQSLAFIRKSSarE 2275.6 59

Sar20 ISQVNEKINQSLAFIRKSDSar 2261.38 100

From the result, sarcosine substitutions of Glu6, Lys7, Asp19, and Glu20

showed minimal disruption in the original binding activity of the C20 peptide, whereas

most of other sarcosine-substituted peptides dramatically lost their binding affinities to

the 5HB. This study reveals essentially the importance of the amide hydrogens in the

backbone of the C20 peptide for maintaining the binding affinity to the 5HB. The loss

of the hydrogen bonds that are necessary for the !-helix in residues 6 and 7 does not

seem to greatly diminish the binding affinity to the 5HB, and may result from



97

unusually high local enthalpic contributions from the neighboring residues at the

hydrophobic interface. While proline has previously served as a beacon inviting

substitution by structure-promoting peptoid residues, this data reveals sarcosine as an

effective screen for substituting peptide residues with any possible peptoid monomers.

Synthesis and characterization of peptomeric C20 analogue

As a proof of concept and to confirm the predictions of the alanine, proline,

and sarcosine scan results experimentally, we incorporated peptoid monomers with

identical side chains to the peptide residues being replaced into the C20 peptide. Since

sarcosine substitutions at Glu6, Lys7, and Glu20 had relatively less impact on the

binding affinity of the C20 to the 5HB, we created an initial peptomer by replacing

with Lys7 and Glu20 with N-(4-aminobutyl)glycine (NLys) and N-(2-

Figure 4-4. % Inhibition of sarcosine-substituted peptides at 100 and 200 µM Each




98

carboxyehtyl)glycine (NGlu) to structurally mimic Lys and Glu, respectively (Fig. 4-

5). Glu6, Lys17, and Asp19 were not replaced with peptoids due to the variation in %

inhibition values. After the synthesis, the peptomer (NLysNGluC20) was purified by

preparative RP-HPLC with > 90% purity.

The binding affinity of this peptomeric C20 analogue to the 5HB was

monitored using our 5HB-based competitive FP assay (Fig. 4-6). The stock solution of

this peptomer was prepared in water in the presence of 30% acetonitrile to reduce the

viscosity and diluted into the FP buffer prior to use. Unfortunately, the binding affinity

to the 5HB significantly decreased down to a % inhibition of only ~ 30 at 200 µM,

presumably due to the additive effects of two substitutions. We are now synthesizing

(A)

NO

O O

NO

NH3

NGlu = N-(2-carboxyethyl) glycine

NLys= N-(4-aminobutyl) glycine

(B)

NH2NNH

HN

NH

HN

NH

HN

NH

HN

NH

HN

NH

HNN

HN

NH

HN

NH

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

HO

OH

NH2

NH

OH

NH2

O

OH

H2NO

HN NH2

H

O

NH2

O

O

NH2O

HN

H2N

O

OOH

OHO

NH2

Figure 4-5. (A) Structures of peptoid residues corresponding to Lys and Glu residues, respectively and (B) structure of NLysNGluC20 peptomer (peptoid residues in shaded in dotted boxes)



99

singly-substituted peptomers with substitutions both at residues predicted as tolerating

peptoid substitutions and at other locations, since such a comparison is a more

appropriate evaluation of the scanning results.

Notably, this is the first example of rationally creating peptomers based on a

data set obtained from a systematic scanning approach. Theoretically, one can imagine

that by replacing amino acids with their counterpart peptoid monomers, proteolytic

degradation could be minimized, thereby counteracting a decreased binding affinity

with a longer circulation half-life.

Figure 4-6. Competitive FP assay of NLysNGluC20 The FP response of Fl-C35 was monitored in the presence of the 5HB as the concentration of NLysNGluC20 increased.



100

Importance of !-helices in biological settings

To find better usage for our combined alanine, proline and sarcosine scans, it is

important to note that peptides derived from the HRB domain such as the C20 peptide

are poorly structured in monomeric form, but upon binding to the innercore HRA

helices, they adopt !-helix conformation.31,32

In general, short peptides are not able to

maintain well-defined 3D-structures in water, implying that there can be an immense

number of conformational isomers present whose loss upon binding is entropically

unfavorable. Thus, the lack of a defined structure in the C20 peptide greatly weakens

its binding affinity (IC50 = 35 µM) to the 5HB compared to longer peptides such as

C35 (Kd = 38 nM) and also leads to high susceptibility to proteolytic degradation.

!-Helices are one of the most abundant structural features of proteins and

many proteins recognize binding partners through contacts with the surface of !-

helices. However, typically the !-helix does not form on its own, and instead the

peptide tends to have a floppy structure, leading to a high entropy penalty associated

with adopting an ordered structure upon binding to the target protein.33,34

To overcome

this unfavorable entropy penalty, numerous approaches to enforce !-helix formation

have been tested including helical capping,35,36

intermolecular cyclization via disulfide

bonds,37-39

lactam bridges between Lys and Asp or Lys and Glu,37-41

Intramolecular

cyclization via click chemistry,42-44

and hydrocarbon stapling.45-49

These efforts have

yielded notable success in increasing biological activities of peptides and overcoming

unfavorable biophysical properties, which has helped revive the prospects of peptides

becoming drug candidates.



101

We thus further utilized the knowledge gained from the combined scan

approach to design C20 peptide analogues that can adopt a stable !-helical

conformation and retain its binding affinity to the 5HB. In this study, two separate

strategies were explored, as discussed below.

Structurally constrained C20 peptide derivatives via hydrocarbon stapling

One method we investigated to stabilize the secondary conformation of the C20

peptide is by hydrocarbon stapling. The incorporation of hydrocarbon-staple links into

peptides at (i, i + 4) or (i, i + 7) positions has been widely investigated in various

fields, yielding great success.45-47

For introducing hydrocarbon stapling into the C20

peptide, two “designer amino acids” which have both an !-methyl group and an !-

alkenyl group (e.g., (S)-2-(4’-pentenyl) alanine) were incorporated at the positions

determined based on the results from the alanine, proline, and sarcosine scanning

studies (Fig. 4-7). Because the efficiency of the stapling reaction depends on the

Figure 4-7. Predicted stabilized helical conformations by hydrocarbon stapling (A) SUMP1 and (B) SUMP2 Non natural amino acids ((S)- 2-(4’-pentenyl) alanine) were incorporated at (i, i + 4) within the C20 peptide, indicating that reactive residues would be

located on the non-binding face of the !-helix.



102

positions in the peptide sequence, it is important to determine the right positions for

the designer amino acids. The resulting two stapled peptides with (i, i + 4) stapling on

the solvent-exposed helix space spanning one turn of the helix were commercially

obtained with > 90% purity.

• SUMP1 (NH2-ISQVNEKINASLAFIXKSDX-NH2, X= hydrocarbon-stapled

positions) was designed to staple one turn of the helix at the C-terminus of the

C20 peptide. This (S)-2-(4’-pentenyl) alanine was incorporated at Arg16 and

Glu20 identified from the alanine and sarcosine substitution studies,

respectively. Unlike SUMP2, the C-terminus of SUMP1 was amidated to

overcome difficulty in the stapling reaction.

• SUMP2 (NH2-ISQVNEKINASLXFIRXSDE-CO2H, X = hydrocarbon stapled

positions) was initially designed to have a helix turn induced by the

hydrocarbon stapling close to the middle of the peptide. Ala13 and Lys17 from

the alanine and alanine/sarcosine substitution studies, respectively, were

chosen for (S)-2-(4’-pentenyl) alanine incorporation.

(A) (B)

Figure 4-8. CD spectra of (A) SUMP1 and (B) SUMP2 The helicity of each stapled peptides were calculated based on CD spectra presented. TFE was used as a helical-promoting solvent.



103

To evaluate if hydrocarbon stapling increases the helicity of the C20 as desired,

the secondary structure of each stapled peptide was monitored by CD spectroscopy

(Fig. 4-8 and Table 4-4). All stock solutions were prepared in 50% acetonitrile in

water for solubility reasons, and were then diluted with an equal volume of either

water (0% TFE) or TFE (50% TFE). The CD spectra indicate that SUMP2 is

significantly more helical in the absence of TFE than SUMP1. As previously reported,

!-helicity tends to be more easily promoted when the hydrocarbon stapling is

introduced in the middle of the peptide.50

Therefore, our observation appears to be

reasonable. We also examined the helical propensity of stapled peptides using

trifluoroethanol (TFE), a well-known helix forming solvent.33

At 50% (v/v) TFE, both

stapled peptides showed extremely helical conformations with over 80% !-helicity.


-1) at 222 and 208, and percent helicity

calculated for stapled peptides and clicked peptomers

Peptides

/Peptomers Solvent [#]222/[#]208 % Helicity

b

0% TFE -a 8

c

C20 50% TFE - 50

0% TFE 0.52 10 SUMP1

50% TFE 0.69 84

0% TFE 0.84 67 SUMP2

50% TFE 0.89 88

a Since the absorbance was too weak, [#]222/[#]208 was not calculated.

b % Helicity was calculated using K2D2

51,52 based on data points ranging from 190 and 240 nm.

c Data points from 200 to 240 nm was used.



104

To investigate if the helical propensity correlates with the binding affinity of

the stapled peptides, the 5HB-based FP assays of SUMP1 and SUMP2 were

performed. Unfortunately, introducing the hydrocarbon stapling into the peptide

significantly lowers its aqueous solubility, particularly for SUMP1. After many

different combinations of solvents (e.g., methanol and acetonitrile) were tested, the

stock solution of SUMP1 still appeared to be very viscous, thereby exhibiting

extremely high polarization values, which makes the FP assay impractical. Efforts to

find a proper solvent system for SUMP1 are on-going. Concentrations ranging from

0.1 to 200 µM of SUMP2 were tested using the 5HB-based competitive FP assays

(Fig. 4-9), demonstrating that SUMP2 (IC50 = 27 µM) could bind to the 5HB slightly

better than the parent C20 peptide (IC50 = 36 µM).

Figure 4-9. Competitive FP assay of SUMP2 The FP response of Fl-C35 was monitored in the presence of the 5HB as the concentration of SUMP2 increased.



105

Overall, SUMP2, which has high helical content either in the presence or

absence of TFE, has a lower IC50 value for binding to the 5HBcompared to C20,

although the increase in the binding affinity was not as dramatic as expected.

Structurally constrained peptomers using click chemistry

The click chemistry method (a coupling of an azide and an alkyne to forma

triazole) has been widely applied to many research disciplines,53

and successfully

improved pharmacokinetic features of peptides (e.g., enhanced bioactivity and

increased protease resistance) by constraining the structures.42-44

In addition, peptoids

have also been subjects for click chemistry, resulting in linear/cyclic peptoids with

multiple clicked functionalities,54-57

and induced helical conformations.58

However,

click chemistry has not been applied to peptomers yet.

Therefore, the second strategy we employed is the use of the click chemistry

method on peptomeric C20 analogues. This approach is expected to achieve enhanced

protease resistance by incorporating two peptoid monomers with clickable functional

groups, i.e., azido and alkyne groups and simultaneously to stabilize the helical

Figure 4-10. Predicted stabilized helical conformation of the C20 peptide using click reaction Nonnatural amino acids with clickable functional groups were incorporated at (i, i + 4) within the C20 peptide, indicating that reactive residues would be located on the safe

face of the !-helix.



106

conformation of the peptomers (Fig. 4-10). Arg16 and Glu20 were chosen based on

the data resulting from the alanine and sarcosine scans, and were replaced with

peptoid residues with azidopropyl and propargyl side chains, respectively (Fig. 4-11).

Since these selected residues are at (i, i + 4) position, it is expected that one-turn of the

helix at the C-terminus of the C20-based peptomer (C20_C) can be formed by click

chemistry cyclization.

To enhance the efficiency of the click reaction, microwave irradiation was

used and all reagents were freshly prepared prior to the experiments. To avoid

multimerized products and possible steric hindrance from the rest of peptide fragment

that might impede the completion of click reaction, the click chemistry was performed

on-resin on partially synthesized peptomer before completion of the peptomer

synthesis.

The completion of the click reaction can be monitored by various methods

including the growth of a new band at !1684!cm"1

in IR (infrared) spectroscopy with,

corresponding to the triazole ring,59

HPLC analysis with retention time (Rt)

difference,43,55

and a modified Kaiser method.60

Since the click reaction was

N

O

N

O

N3

Azidopropyl side chain Propargyl side chain

Figure 4-11. Structures of peptoid residues with clickable functional groups



107

performed on resin, IR spectroscopy would not have been effective, and thus we

decided to use a modified Kaiser method, which utilizes triphenyphospine (TPP) to

reduce unreacted azide to amine and ninhydrin to detect free amine group (see

Materials and methods). Following the completion of the click reaction between the

two peptoid monomers, the synthesis of rest of the peptide sequence was completed

and the clicked peptomer was purified by preparative RP-HPLC with > 90% purity,

yielding C20_C_C (Fig. 4-12B). Unclicked peptomer was also prepared as a control

(C20_C_U) (Fig. 4-12A).

Figure 4-12. Structures of (A) C20_C_U and (B) C20_C_C with desired molecular weights Azide and alkyne groups are presented in red and blue, respectively. The molecular weight of each peptomer is the same, suggesting additional method is required o confirm the completion of the click reaction and the synthesis.



108

To examine if the clicked peptomer could demonstrate increased helical

content, the secondary structure of each peptomer was analyzed by CD spectroscopy.

The CD spectra indicated that both C20_C_C and C20_C_U were poorly helical in

water with 25% (v/v) AcN, exhibiting [#]222 values of approximately -5,000

deg$cm2$dmol

-1 and insignificant % helicity (Fig. 4-13 and Table 4-5). The helical

propensity of each peptomer in 50% (v/v) TFE was also studied, suggesting that the

clicked C20_C_C showed higher helical content than its unclicked analogue, C20_C_U

and C20, which confirms that our attempt to stabilize peptomer secondary structure

using the click chemistry was successful.

A) (B)

Figure 4-13. CD spectra of (A) C20_C_U and (B) C20_C_C The helicity of each peptomer were calculated based on CD spectra presented. TFE was used as a helical-promoting solvent to determine how easily the helical conformation of the stapled peptides can be promoted.



109


-1) at 222 and 208, and percent helicity

calculated for stapled peptides and clicked peptomers

Peptides

/Peptomers Solvent [#]222/[#]208 % Helicity

a

0% TFE - 8 C20 50% TFE - 50

0% TFE 0.60 8 C20_C_U

50% TFE 0.76 67

0% TFE 0.56 8 C20_C_C

50% TFE 0.77 78

a % Helicity was calculated using K2D2

51,52 based on data points ranging from 190 and 240 nm.

To evaluate the correlation between the helical propensity and the binding

affinity of the clicked and unclicked peptomers, the 5HB-based competitive FP assays

were carried out using C20_C_C and C20_C_U. Although the clicked C20_C_C, which

has the highest helical content in 50% (v/v) TFE, seemed to bind to the 5HB less

tightly compared to the C20, C20_C_C exhibited slightly higher binding affinity to the

Figure 4-14. Competitive FP assay of C20_C_U and C20_C_C The FP response of Fl-C35 was monitored as the concentrations of each peptomer increased.



110

5HB than its unclicked analogue, C20_C_U (Fig. 4-14).

Although the helix-stabilization strategy using click chemistry on peptomers

seemed effective given the CD spectroscopy results, it is possible that the highly

structured peptomers might be in an inactive conformation, and thereby have poor

binding affinity to the 5HB. It is also well-known that in many cases introducing helix

constraints in the middle of a helix instead of near the terminus is more effective, and

we are in the process of synthesizing these derivatives.



111

Conclusion and future prospects

Here we propose a generally applicable method using alanine, proline, and

sarcosine for determining peptoid-replaceable peptide residues and attempt to prove

the uniqueness and usefulness of the combined scan using C20 as a model peptide. This

combined approach has generated useful data sets related to the conformational

tolerance and importance of individual side chains, which cannot be easily provided

by structural studies such as an X-ray crystallography.

Figure 4-15. Helical wheel representation of the C20 peptide C20 peptide is presented as a helical wheel projection and peptoid-replaceable peptide residues are in red circle. The positions for hydrocarbon stapling and click reaction were connected with dotted line. The top view of the 5HB is included, showing three inner HRA helices in green and two outer HRB helices in red.



112

The results we obtained through this approach are summarized in Fig. 4-15,

implying that peptide residues pointing to the binding interface are crucial to retain the

binding affinity, while amino acids facing away from the binding site are better

candidates for peptoid replacement and further structural modifications. A prototype

peptomer, NLysNgluC20, were first tested, proving that it is plausible to predict

peptoid-replaceable peptide residues in the target peptide sequence without

transforming each peptide residue to the corresponding peptoid monomer

experimentally. Furthermore, we investigated two different strategies to promote the

!-helical conformation of the C20 peptide via either hydrocarbon stapling between

nonnatural amino acids with olefinic side chain or click chemistry cyclization between

clickable peptoid monomers. The structurally constrained C20 analogue, SUMP2, was

more helical and exhibited increased binding affinity to the 5HB as compared to C20.

Although another conformationally restricted C20 analogue, the clicked

C20_C_Cshows helicity that is clearly improved by the click reaction, its binding

affinity to the 5HB was not improved (Fig. 4-16).

Figure 4-16. Comparison of % Inhibition and helical propensity of structurally constrained peptomer (C20_C_C) and peptide (SUMP2) are presented in comparison with

those of C20. %Inhibition was calculated at 200 µM of each compound and for helical

propensity, per residue molar ellipticity at 222nm was measured in 50%(v/v) TFE.



113

Our findings suggest that it is helpful but apparently not sufficient in all cases

to have high helical propensity (i.e., how easily the helix can be promoted).

Additionally, we recognize that the carbon chain formed by the click reaction in our

study is one carbon shorter than the hydrocarbon stapling approach, which might

interfere with the ability of the peptomer to adopt a properly formed !-helical

conformation.

Our system, which predominantly consists of an !-helix, is suboptimal in

proving the true value of the combined scanning approach to develop peptidomimetics.

Protein interactions through !-helices require a precise spacing between peptide

residues from each binding partner, with less than ideal tolerance of incorporating

peptoids into the peptide sequences (e.g., the conformational difference of the peptoid

helix and !-helix). Beyond the immediate application to the hRSV system, we hope

that our novel strategy presented here will be more useful and powerful on other

systems (e.g., "-turn mimics and miniature protein scaffolds) and provide a critical

design paradigm for future therapeutic peptidomimetics.



114


General materials

Reagents for peptide and peptomer synthesis were purchased from Applied

Biosystems (Foster city, CA, USA) or Sigma-Aldrich (Milwaukee, WI, USA). Resins

and Fmoc-protected amino acids were purchased from NovaBioChem (San Diego,

CA, USA) or Anaspec (San Jose, CA, USA). Solvents and reagents for peptoid

monomer synthesis and for analytical and preparation RP-HPLC were purchased from

Fisher Scientific (Pittsburgh, PA, USA). All other chemicals were purchased from

Sigma-Aldrich (Milwaukee, WI, USA) and used without additional purification.

Alanine/proline/sarcosine substituted peptides synthesis

Peptides with alanine, proline, and sarcosine substitution were commercially obtained

with 95% purity (Aapptec, Louisville, KY, USA) and the sequences are listed in Table

4-1, 2, and 3. Final purities and the molecular weight of synthetic peptides were

conrimed by Aapptec and analytical HPLC traces and data sheet of electrospray mass

spectrometry (ESI) were also provided.

Stock solution preparation

In the case of using dimethyl sulfoxide (DMSO) for the peptide stock solution in a

high concentration (e.g., 5 mM), immediate use is recommended. At room temperature,

peptide stock solutions in DMSO appeared to be clear with no sign of precipitation.

However, after being stored at 4 °C, the solutions may become frozen due to low

melting point of DMSO (18.5 °C) and stay turbid and viscous, even after being



115

incubated at room temperature over 1 hr. This will interfere with polarization

measurement. To overcome this solubility issue and enable to store the solutions at 4

(~ days) or -20 °C (~ weeks), 50% of acetonitrile is recommended.

Sample preparation for FP measurements

Few peptides were extremely insoluble compared to other peptides. Because the

testing concentrations of alanine/proline/sarcosine-substituted peptides for the 5HB-

based competitive FP assays were relatively high (100 and 200 µM), precipitations

were often observed. To reduce loss of peptides due to their poor solubility, vortexing

and/or sonication were carried out. In addition, to minimize undesirable perturbation

in polarization measurement caused by remaining precipitates, the solution of each

sample was spun down and then transferred to the 96-well plate with care, followed by

10 sec delay before measurements.

Stapled peptides preparation

Stapled peptides were synthetically prepared with 90% purity (Anaspec, San Jose, CA,

USA). The peptide residues for hydrocarbon stapling were chosen based on the results

from our alanine/proline/sarcosine scans and the synthesis of hydrocarbon-stapled

peptides was carried out as previously reported.45-47

Final purities and the molecular

weight of synthetic peptides were confirmed by provided analytical HPLC traces and

data sheet of ESI.



116

Monomer preparation: 3-Azidopropylamine61

Synthesis of 3-azidopropylamine (2) is shown in Scheme 1. Commercially available 3-

chloropropylamine (1) is refluxed with 3 eq. of sodium azide in water at 80 °C

overnight to complete the conversion of (1) to (2). The reaction was then cooled and

treated with NaOH (10 M) until pH became basic. The aqueous solution was extracted

using chilled ethyl ether three times and then the combined organic layer was dried

over Na2SO4, and filtered. Due to the low boiling point of the desired product, the

organic solution was concentrated under weak N2 flow for over 1 hr. Resulting 3-

azidopropylamine and H1 NMR confirmed that (2) was obtained with reasonable

purity. After concentration was determined, (2) was diluted in N-Methylpyrrolidone

(NMP) as a final concentration of 1.0 M for peptoid synthesis. 1H NMR was used to

determine the concentration of the solution; solvent was further evaporated if

necessary. 1H NMR (500 MHz, CD3OD) # 3.40 (t, J = 7.0, 2H, N3CH2), 2.79 (t, J =

7.0, 2H, H2NCH2), 1.77 (m, J = 7.0, 2H, CH2CH2CH2). 1H NMR of 3-

azidopropylamine was performed by Dr. Sungyoung Seo (Stanford University).

Cl NH2 N3 NH2i) NaN3, 80 °C

ii) NaOH1 2

Scheme 1. Synthesis of 3-azidopropylamine



117

Synthesis of peptomeric C20 analogue

Peptomeric C20 analogue, NLysNGluC20, was synthesized in the laboratory using

standard Fmoc chemistry for peptide residues and a submonomer peptoid synthesis62

with peptoid monomers using an automated ABI 433A peptide synthesizer (Applied

Biosystems, Foster city, CA, USA). After synthesis, the peptides were cleaved off

from the resin and deprotected in trifluoroacetic acid (TFA)/water/TIPS/thionisole

(90:5:2.5:2.5 v/v) for 1.5 hr at room temperature. Peptomers were purified by

preparation RP-HPLC on a C18 column using a linear gradient of 5-99% solvent B in


(v/v) TFA in acetonitrile). Final purities of synthetic peptides were confirmed to be >

95% by analytical RP-HPLC, and the molecular weight of the purified product was

confirmed by ESI at the Stanford University Mass Spectrometry (SUMS) facility.

Synthesis of peptomeric C20 analogues for click reaction

Peptomeric C20 analogues (C20_C_C and C20_C_U) were synthesized in the laboratory

as described above. For an unclicked peptomer as a control, the synthesis is completed

without any interruption. On the other hand, for a clicked peptomer, microwave-

assisted click chemistry was carried out after first 6 residues including peptoid

monomers were synthesized. The synthesis for the rest of the C20 peptide was then

completed. The cleavage and purification of these peptomers were done as described

above.



118

Microwave-assisted click chemistry

Click chemistry (1,3-dipolar cycloaddition) between the azide and alkyn

functional groups was performed on solid support (rink amide resin). Stock solutions

were prepared: (i) 1 M of CuSO4$5H2O (Santa Cruz Biotechnology, Santa Cruz, CA,

USA) was dissolved in H2O; (ii) 1M of L-(+)-sodium ascorbate (Santa Cruz

Biotechnology, Santa Cruz, CA, USA) was dissolved in t-BuOH/H2O (1:1). For a

microwave-assisted reaction, 0.13 mmole of partially synthesized peptide-bound resin

in 75 mL of t-BuOH/H2O (1:1) was placed in the reaction tube and subsequently L-

(+)-sodium ascorbate (520 µmol) and CuSO4$5H2O (130 µmol) were added. The

reaction tube was sealed and heated in the microwave reactor for 1 hr (70 °C,

absorption level: high). The reaction mixture was pale yellow after microwave heating.

The clicked and unclicked peptomers were purified by preparation RP-HPLC on a C18

column using a linear gradient of 5-99% solvent B in solvent A over 60 min (solvent

A is 0.1% (v/v) TFA in water and solvent B is 0.1% (v/v) TFA in acetonitrile).

NHNO

N3

NH

HN N

O

O

OO

O

ONH

OHN

O

NH

Fmoc

Prt

Prt

Prt

NHNO

NH

HN N

O

O

OO

O

ONH

OHN

O

NH

Fmoc

Prt

Prt

Prt

Cu(II), Ascorbate

microwave, 70 °C, 1 hr

NN

N

Scheme 2. Microwave-assisted Cu (II) catalyzed click chemistry on resin Azido and alkyne functional groups in peptoid side chains are shown in red and blue, respectively. To enhance the reaction efficiency of the click chemistry, on-resin click chemistry is adopted in this study.



119

As shown below, the resulting clicked peptomer (Rt = 16 min) was eluted

faster then the unclicked one (Rt = 18 min) analyzed by analytical RP-HPLC on a C18

column using a linear gradient of 5-99% solvent B in solvent A over 30 min (solvent

A is 0.1% (v/v) TFA in water and solvent B is 0.1% (v/v) TFA in acetonitrile).

Comparison of the analytical HPLC traces of purified C20_C_C and C20_C_U

Modified Kaiser method

To verify the completion of the click chemistry, a simple colorimetric method based

on Kaiser test was carried out.60

200 µL of 5% triphenylphosphine (TPP) was added

on to the peptomer-bound resin and then the solution were vortexed, followed by the

addition of 5% ninhydrin in ethanol. The mixture was heated at 130 °C till the strong

colors showed up (dark purple). Unclicked peptide-bound resin and resin only were



120

also tested as controls. For best results, it is highly recommended that TPP solutions

should be prepared prior to use and/or stored under inert atmosphere to minimize air

oxidation.

Scheme 3. Modified Kaiser method to determine the completion of the click reaction

Competitive fluorescence polarization (FP) binding assay

Competitive FP binding assays of the C20 derivatives were carried out as previously

descibed.63

Briefly, using a 5HB protein construct and a fluorescently labeled 35

amino acid-long peptide (Fl-C35) as a target and a tracer respectively, the binding

affinity of each C20 analogue prepared in this study was individually evaluated. Each

well in a black 96-well plate (Corning Inc. Lowell, MA, USA) contained 20 nM of the



121

5HB and increasing concentrations of each peptomer in FP buffer (20 mM PBS at pH

7.4, 500 mM NaCl, 0.01% (v/v) TweeN-20, and 0.05 mg/ml bovine gamma globulin)

in a final volume of 185 µL with 1hr incubation at room temperature. Fl-C35 was then

added to a final concentration of 5 nM followed by 30 min incubation at room

temperature. The FP responses were monitored using a Synergy4 plate reader (Biotek,

Winooski, VT, USA) with an excitation wavelength of 485 nm and an emission

wavelength of 530 nm. The percentage of inhibition (% Inhibition) was calculated

using the following equation: % inhibition = 100%[(mP-mPf)/(mPb-mPf)], where

unbound Fl-C35 (mPf), bound Fl-C35 (mPb) and the bound inhibitor to the 5HB (mP)

are used accordingly.


CD spectra were obtained with a Jasco J-815 spectrophotometer (JASCO,

Easton, MD, USA). Sample was prepared in water with 25% (v/v) acetonitrile and

50% TFE for studying the helical promotion potentially induced by hydrocarbon

stapling and click reaction. The concentrations ranging from 25 – 50 µM were used.

Data were recorded from 190 to 260 nm with a step size of 0.2 nm, at a rate of 100

nm/min, a bandwidth of 1.0 nm with a scanning speed of 20 nm/min in a 0.1 cm path-

length quartz cell. The response time was 0.5 s. Each CD spectrum was an average of

3 measurements and the baseline was corrected by subtracting the spectrum of a blank

obtained under identical conditions. The resulting data was converted to per-residue

molar ellipticity units (deg·cm2·dmol

-1·residue

-1) and the secondary structure content

was analyzed with a web-based software, K2D2.51,52



122

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Figure 5-1. Schematic diagram of a filamentous phage (M13) Coat proteins are “displayed” on pIII or pVIII.

Chapter Five

Identifying short peptide-based hRSV entry inhibitors from a phage-

displayed peptide library and their peptidomimetic analogues

In previous chapters we discussed our knowledge-based approaches to the

identification and optimization of peptide and peptomer therapeutic candidates. These

candidates were all guided by the known sequence and structure of the hRSV F

protein 6-Helix Bundle. To ensure that we had left “no stone unturned,” we wanted to

complement our knowledge-based approaches with unbiased library screens, and

therefore turned to phage displayed peptide libraries.

Overview of bacteriophages

Bacteriophages, or simply phages, are

single-stranded DNA viruses that infect a

variety of Gram-negative bacteria using pili as

receptors.1 Various engineered phage particles

have been intensively studied for medical and

therapeutic applications as well as for the

development of new materials and

nanostructures.2-4

As a research tool, the

filamentous phage, M13, is one of the most

Chapter Five: Short peptide-based hRSV entry inhibitors and their peptidomimetic analogues

130

widely used phages, because it contains genetically easily modifiable genes for coat

proteins, which are useful and versatile for display purposes. The M13 genome

consists of 11 genes, expressing about 2,700 copies of the pVIII coat protein and 3 to

5 copies of the pIII coat protein (Fig. 5-1).1,5

Although infection with M13 phages is

not lethal to bacterial cells because it is a non-lytic virus (i.e., M13 phages neither lyse

nor destroy the bacterial cells after replication), the rate of cell growth is detrimentally

affected when M13 phages infect bacteria.

Phage display technology

Phage display was first introduced in 1985 as an effective method to map

epitope-binding sites of antibodies by screening phage-displayed peptide libraries

against an immobilized immunoglobulin.6 The basic principle of phage display is to

literally “display” desirable peptides or proteins on the surface of the phage. After

exposure to a target protein, specific clones of phages displaying peptides that bind to

the target protein can be easily selected and amplified. To construct the phage-

displayed peptide libraries, DNA sequences of interest with variable regions are

inserted in the genome of the phage such that the encoded protein is expressed as a

fusion product to one of the phage coat proteins (e.g., pIII). Therefore, several billion

peptide variants can be constructed simultaneously instead of modifying individual

genetic material, expressing, purifying and analyzing each variant one-by-one.1 The

advantage of phage-displayed peptide libraries over chemically synthesized peptide

libraries is that the physical connection between the surface-displayed peptides and the


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internal genetic coding allows successive rounds of optimization to be carried out

without regaining laborious sequencing and identification at each round.

The length of the peptides displayed on phages was historically limited to 6 –

15 residues, due to the possible interference with the coat proteins’ endogenous

function and poor display efficiency. However, this limitation has been overcome by

using a helper phage or encoding an additional copy of coat protein genes into a

phagemid vector.7 The sequences of the peptides of interest can be randomly or

rationally designed to increase the odds of obtaining optimal sequences (e.g., by fixing

positions of certain amino acids or by introducing structural constraints such as

cyclization of the peptides via disulfide bond).8 Typical phage-displayed library sizes

range from 107 to 10

9 of individual transformants, though libraries up to 10

11 have

been produced.9

Once the library is obtained, the phage particles are exposed to the target

proteins in an immobilized or solubilized form to identify specific ligands or enrich

previously selected ligands with enhanced binding affinity. After non-specific binders

are removed, the phages bound to the target are recovered and subsequently amplified

for further rounds of the selection. This in vitro selection process called “panning” is

typically repeated for 3 – 5 rounds until enrichment of the specific phages is achieved

(Fig. 5-2).9 After the panning, DNA is extracted from individual phages and

sequenced to identify the protein phenotype of each clone.


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Figure 5-2. Summary for obtaining high-affinity peptide ligand for binding to target molecules using phage-displayed peptide libraries

When the peptide binders are enriched and their sequences are revealed, the

consensus sequence among the identified peptides are often found, which are

considered as more important since they represent a “convergent evolution” solution

that likely serves as the optimized endpoint of multiple initial designs. For this

purpose, the consensus sequences can serve as a useful scaffold for building more

focused libraries for affinity maturation, if necessary.10

Once the peptides with desired

properties including high selectivity and biological activity are chosen, synthetic

peptides are prepared and then further investigated to obtain insight on the

relationships between their structures and biological functions, which can be used to

design peptide, non-peptide, or peptidomimetic ligands.


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Applications of phage display technology to antiviral development

Since the first phage display library was produced,6 phage display technology

has become a powerful approach to discover therapeutically useful peptides and

improve their biophysical properties as suitable pharmaceuticals. Many novel peptides

identified by phage display are now in clinical trials as therapeutics to treat a variety of

diseases are used as diagnostic tools in various biological and medical research areas,

including the antibody identification,11

enzyme inhibitors,12,13

gene delivery,14,15

cancer targeting,16-19

protein-binding peptides,20-22

DNA-binding peptides,23

and

tissue-specific peptides.24

Specifically, considerable efforts have been made to identify

antiviral peptides targeting various viruses. For instance, antiviral peptides targeting

West Nile virus (WNV) envelope protein (E) have been isolated from phage-displayed

peptide libraries. The E protein is known to mediate virus attachment to the host cells

and subsequent viral fusion with the host cell membrane, is therefore an attractive

therapeutic target. Screening of phage-displayed peptides and proteins derived from

murine brain cDNAs against the WNV E protein yielded several short peptides

ranging from 8 to 26 amino acid in length with the potential of crossing the mouse

blood-brain barrier.21

HIV-1 gp120 bound peptides were also identified from three

randomly designed peptide libraries. The functional properties of the identified

dodecameric peptides were investigated, suggesting that they would stabilize HIV-1

gp120 conformations and might be useful as leads for designing HIV-1 entry

inhibitors.20

Another interesting application of phage display is mirror-image phage

display, which has yielded successful D-peptide ligands for diagnostic and therapeutic


134

applications.25

There is a growing interest in the therapeutic usage of D-peptides,

particularly because they are highly protease-resistant and thereby present prolonged

in vivo half-lives compared to L-peptides. Briefly, the strategy employed for the

mirror-image phage display begins with a synthetically generated therapeutic D-

enantiomeric form of the target protein followed by screening the phage-displayed L-

peptide library against them. After identification of efficacious L-peptides against the

D-target, equivalent D-peptides are synthesized for use against the actual biological L-

target (Fig. 5-3). Specifically, the D-peptide HIV-1 entry inhibitors targeting HIV-1

gp41 have been identified using mirror-image phage display library with high potency

(IC50 = 250 pM) and binding affinity to the natural protein targets.26-29

Figure 5-3. Concept of mirror-image phage display The D-enantiomeric form of target peptides are used for phage display. Once L-enantiomeric peptides bound to the D-enantiomeric targets are isolated, the D-enantiomeric form of selected L-peptides can be synthesized, providing enzymatically stable peptides


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hRSV F protein as a target for phage-displayed peptide library

Although phage display has allowed for the rapid and cost-effective screening

of a huge number of peptide libraries,9 attempts to identify antiviral peptides to control

hRSV infection using phage display libraries have not been pursued yet. Previously,

we reported that we can easily screen potential hRSV entry inhibitors using a protein-

based fluorescence polarization (FP) assay, which is scalable to a high-throughput

screening (HTS) format (see the details in Chapter 2).30

hRSV F protein-mediated

viral fusion is the most essential step of the hRSV infectious cycle. As discussed in

Chapter 1, this viral fusion occurs extracellularly, and is therefore an attractive drug

target because of its relatively easy access to antivirals.

In this study, we applied the Ph.D.!-12 phage display peptide library (New

England Biolabs, Ipswich, MA, USA) to an engineered hRSV F protein (5-Helix

Bundle, 5HB) to identify peptide-based inhibitor candidates to block hRSV F-

mediated viral fusion. After isolating phage-bound peptides that specifically bind to

the target (5HB), peptides were individually synthesized and their binding affinities to

the 5HB were examined using the 5HB-based FP assay. Even with the short length (12

amino acid-long), which might cause a poorly structured conformation and high

susceptibility to proteolytic degradation, the selected peptides showed modest binding

affinities to the 5HB. To reduce the protease susceptibility of these peptides, peptoid-

peptide hybrids were synthesized and tested. Furthermore, to understand the

interaction of the identified peptides with the 5HB, the effort of co-crystallization of

these peptides with the 5HB is being pursued and is expected to provide insight into


136

how these short peptides can bind to the 5HB and thus provide a design basis for

peptidomimetic antivirals.

Phage-displayed peptide library panning results and ELISA tests

With the aim of isolating peptides that specifically bind a hydrophobic groove

where a missing 6th helix of hRSV F protein 6-helix bundle (6HB) binds, the 5HB was

used as a target during the panning process.30

Random 12-mer peptides from Ph.D.!-

12 phage display peptide library with a capacity of ~ 2.7 x 109 sequences were tested

against the 5HB directly. After 3 rounds of the panning, the DNA extracted from 30

plaques of individual phage clones was sequenced, resulting in 16 unique phage clones

and 2 wild type phages (pIII protein only). The sequences and their frequency of

isolation during the panning are shown in Table 5-1.

Table 5-1. Sequences of phage-bound peptides isolated by screening against the 5HB

16 different peptide sequences were identified.

aID

Sequence

(amino to carboxy)

Frequency

of isolates

P1-2 WHWSWQPQRHSP 3

P1-5 WHWSVPWAPLHE 1

P1-7 VAAPAKATMSST 3

P1-8 THKYANYQWQPR 4

P3-1 WHWFPTAPSYRA 1

P3-4 FPQMHNGPSTRT 2

P3-8 WHWQPYVPWTPR 1

P4-1 KCCYPDIQPNSR 3

ID Sequence

(amino to carboxy)

Frequency

of isolates

P4-2 VLAAPSISHRTL 1

P4-3 HLHALSSLPTPL 1

P4-6 KLHQRVMPTPLW 1

P4-7 DARIMPRPLGPY 2

P4-9 KVWLPHNPTLNI 1

P5-1 GLKIWSLPPHHG 2

P5-3 QPIKVMPMGWAT 1

P5-7 RCCHPNVPEISA 1

aEach clone is named by the plate number that it was selected followed by the clone number.


137

Figure 5-4. Binding of selected phage-bound peptides to immobilized 5HB Binding abilities of phage-bound peptides identified from the panning process were measured by using the ELISA assay. BSA was used as a control. The data present mean values of duplicate measurements.

To further assess the selectivity and specificity toward the 5HB, the peptide-

bound phages were amplified and then tested using the ELISA methods. Serially

diluted peptide-bound phages ranging from 104 to 10

11 pfu/mL were incubated in the

5HB- and BSA-coated wells, respectively. BSA was used as a negative control to

prevent isolating non-specific binding peptides.31

The ELISA tests with the 5HB

resulted in four phage-bound peptides with significantly higher binding affinity to the

5HB compared to BSA (Fig. 5-4), corresponding to the sequences

WHWSWQPQRHSP (P1-2), WHWSVPWAPLHE (P1-5), VAAPAKATMSST (P1-

7), and THKYANYQWQPR (P1-8). During the panning and ELISA experiments, the

protein-coated wells were filled with a blocking solution to the top of each well to

avoid the possibility of isolating plastic-binding phages.31

However, we still observed


138

a high frequency of aromatic residues such as Trp in the identified peptide sequences.

Although the enrichment of aromatic residues could be an indication of plastic-binding

sequences, the abundance of these residues should not be considered as an absolute

diagnostic of plastic binding since there are many reports on the specific binding of

aromatic residue-rich peptides to target proteins such as HIV-1 gp120, and the

hydrophobic binding pocket of the 5HB may be a target as well (see Chapter 2).20,32

Competitive FP assays of selected synthetic peptides

Even with numerous benefits gained from phage display, there are a number of

technical limitations in the panning process: first, the elution conditions of selectively

bound phages should be optimized because overly stringent conditions would reduce

yields of candidate peptides in early stages.33

Second, the avidity effect may lead to

the selection of tight-binding peptides in the context of the phage virion that may not

bind with high-affinity in the context of synthesized monomeric peptides.34

The

avidity due to the polyvalent display magnifies an intrinsic affinity to the target, so

that even weak-binding peptides with poor affinity can bind to the target,10

which

leads to unnecessarily widening of the pool of potential candidates. To eliminate these

intrinsic limiting factors in using the phage display libraries: we first progressively

increased the elution stringency as the panning progressed. Second, to minimize

multivalent avidity effects, we prepared synthetic peptides with 95% purity (Table 5-

2) and further tested the peptides. The 5HB-based FP assay here served as a secondary

assay to select the monomeric peptide ligands with high specificity and also enabled


139

efficient quantification of binding affinity of peptides identified from the phage-

displayed peptide library.

Table 5-2. Peptides individually synthesized and used in this study Proline residues are

in bold and similar sequences found in at least two peptides are underlined.

ID Sequence (amino to carboxy) Molecular

weight (Da) pI

a IC50

P1-8 THKYANYQWQPR 1591.5 9.70 138

P1-2 WHWSWQPQRHSP 1631.7 9.76 NMb

P1-5 WHWSVPWAPLHE 1544.7 6.02 27

P1-7 VAAPAKATMSST 1134.3 8.72 NMb

P5-3 QPIKVMPMGWAT 1358.6 8.75 NMb

apI (Isoelectric point) was predicted with the ProtParam algorithm (http://expasy.org/tools/

protparam.html). b

NM = Non measurable

Each synthetic peptide’s ability to prevent the missing 6th

helix (fluorescently

labeled C35 peptide probe, Fl-C35) binding to the 5HB was determined at the

concentrations of 100 and 200 µM,(Fig. 5-5).30

Only two peptides (P1-5 and P1-8)

showed good % inhibition for Fl-C35 binding to the 5HB, showing an IC50 value of 27

and 138 µM, respectively (Table 5-2 and Fig, 5-6). On the other hand, the other

peptides substantially lost their binding affinities (Table 5-2). As briefly discussed

above, it is not surprising that some of the synthetic peptides showed weaker binding

activities than those of peptide-bound phages, because of high local concentrations of

the peptides due to the avidity effect of polyvalently expressed peptides on the

phage.34


140

Figure 5-5. % Inhibition of Fl-C35 binding to the 5HB by resynthesized peptides using a competitive FP assay A polarization value of 5HB·Fl-C35 in the absence of inhibitors serve as 0% inhibition and unbound Fl-C35 was used as a 100% inhibition value. The polarization

values obtained in the presence of inhibitors at 100 and 200 µM of concentrations were

normalized based on the polarization values of 0 and 100% inhibition.

Figure 5-6. Competitive FP assays of P1-5 and P1-8 The binding affinities of two 12-mer peptides, P1-5 and P1-8 were fully evaluated using the 5HB-based competitive FP assays.


141

Secondary structure analysis of selected synthetic peptides

Since these peptides could displace Fl-C35 for the binding site of the 5HB with

a low micromolar binding affinity, we speculated that the peptides should bind to a

hydrophobic groove formed by two neighboring HRA helices within the 5HB.

However, no significant sequence homology was found between these selected 12-mer

peptides and hRSV F protein or even among selected peptides. We thus pursued the

structural studies on selected synthetic peptides. Even with no obvious sequence

consensus between the different peptides, it is noteworthy that proline residues are

frequently found in identified peptide sequences (Table 5-2). Particularly, P1-5

contains two prolines with a polyproline II (PPII) structure type sequence motif

(PXXP: P = proline and X = other amino acids) suggesting that P1-5 might have a

PPII helical structure. This PPII is typically a short left-handed helix with

characteristic CD spectral features of a negative band at around 200 – 210 nm and a

weaker positive band at 225 – 235 nm (Fig. 5-7A).35

It is also known to play an

important role in hydrophobic interactions and is commonly found in many protein-

protein interactions including SH3 and WW domains.36,37

Because the PPII helix

contains no intramolecular H-bonds, its structure can be more open and flexible than

"-helices, allowing the conformation to easily adopt an appropriate structure when it

needs to bind to any target proteins.38,39

To test if P1-5 or other selected peptides have the PPII type helical structure,

the secondary structure of P1-5 was examined by CD spectroscopy. The CD spectrum

of P1-8 peptide with one proline was also analyzed due to its promising binding

affinity to the 5HB. P1-5 did not present a strong indication as the PPII helix


142

signature, which can be easily confused with random coil (unstructured or unordered)

conformations,39

and the spectrum of P1-8 was a too weak to be assigned as any

distinctively structured conformation.

(A) (B)

Figure 5-7. CD spectra of (A) reference spectra of secondary structures and (B) P1-5

and P1-8 In the reference spectra, "-helical ("), #-sheet (#), random-coil (r), and polyproline

type II (PPII) conformations in green solid line are indicated (adapted from Rath et al. (2005) Biopolymers 80, 179). The characteristic bands of PPII at a negative and a positive peak are marked in dotted and solid line, respectively in both graphs.

Efforts to cocrystallize selected peptides with the 5HB

To better understand how selected peptides form a surface contact with the

target (5HB), co-crystallization or NMR spectroscopy can be useful. The 3-D

structural information can be further combined with functional studies on the selected

peptides, potentially providing valuable insights for more focused libraries for affinity

maturation. We therefore decided to investigate the interaction of P1-5 and P1-8 with

the 5HB using X-ray crystallography. Our initial aim for structural studies was to

obtain co-crystals of these peptides with the 5HB. After more than 300 crystallization


143

conditions were screened, the crystallization effort was still not as fruitful as desired.

In examining related hRSV F protein constructs that successfully yielded the

diffracting crystals leading to structural information,26,40,41

we noted that other protein

constructs are composed of individually chemically synthesized HRA and HRB

peptides. Since the 5HB is a single-chain polypeptide generated by alternatively

connecting three HRA and two HRB helices using short peptide linkers, these peptide

linkers might be placed differently depending on how the helices align in the bundle.

Theoretically, both right-turning and left-turning bundles can occur equally with our

construct, which may provide two different configurations of the 5HB (Fig. 5-8). The

presence of more than one 5HB conformation might explain the difficulties in co-

crystallizing the 5HB with selected peptides.

Figure 5-8. Two different handedness of the 5HB bundling The right- and left-turning 5HB can be formed equally with unwanted heterogeneity, which may interfere with the 5HB

crystallization. (Linker 1 in green: PPPELGGP, Linker 2 in red: KGSSK)


144

Alternative ways to cocrystallize selected peptides with the GCN construct

To obtain a structurally homogeneous protein construct, the crystallization

strategy was thus changed to create a simple construct mimicking the innercore HRA

helices of the hRSV F 6HB. Previously, it has been shown that the HRA domains

easily aggregate in the absence of the HRB domains, due to the hydrophobic groove

presented on the HRA helices.42

The GCN domain was adapted to stabilize the

trimerization and solubilize the protein constructs, thereby preventing the possible

uncontrolled aggregation of the HRA helices (Fig. 5-9).26,43

The GCN domain is 32

amino acid-long (ARMKQIEDKIEEILSKIYHIENEIARIKKLIGEA) and a parallel

trimer-forming domain with high thermal stability (Tm > 100 °C). Two constructs

were designed to consist of the HRA helix fused to the GCN domain both N- and C-

terminally (GHG) or C-terminally only (HG). These constructs were expected to yield

soluble and stable trimerized HRA helices, providing a sufficient binding site to

peptides of interest (Fig. 5-9).

(A) (B)

Figure 5-9. (A) Schematic diagram of GCN constructs (HG and GHG) for crystallization study and (B) illustration of trimerized GCN constructs (arrowhead in red represents TEV protease cleavage site for His8-tag removal)


145

Purified GCN constructs were tested using the 5HB-based FP assay with Fl-

C35 to determine if the binding site is exposed as designed. As shown in Fig. 5-10, Fl-

C35 seemed to bind to both GCN constructs reasonably well, but a difference in

increased polarization values was observed. This can be explained by the following

reasons: first, to have Fl-C35 tightly bind to the GCN constructs, the binding site needs

to be well formed when Fl-C35 is introduced. The GHG construct thus is expected to

be more stable due to two GCN domains being attached to both the N- and C-terminus

of the HRA domain. Therefore, the higher polarization value from Fl-C35 binding to

the GHG construct is reasonable. Second, the molecular weight of the GHG construct

(16.2 KDa when trimerized) is higher than that of the HG construct (12.6 KDa when

trimerized). In FP, a larger target protein exhibits a larger increase in polarization

value upon a probe binding to the target due to slower tumbling rate. Therefore it is

understandable that the GHG construct showed higher polarization value than the HG

construct in the presence of Fl-C35.

Figure 5-10. FP response of GCN constructs in the presence of Fl-C35 peptide 5 nM of Fl-

C35 was used and 30 µM of C35 peptide was included for a positive control.


146

The competitive FP assays of Fl-C35 binding to the GCN constructs in the

presence of a unlabeled C35 peptide at 30 µM were also tested (Fig. 5-10). Even with

an excess amount of the unlabeled C35 peptide (IC50 = 38 nM, see Chapter 2 for

details) used, we observed only ~ 80% decrease in the polarization value compared to

the GCN constructs in the absence of Fl-C35, for both GHG and HG. Since the GCN

constructs theoretically expose three binding sites, it might be difficult to displace all

Fl-C35 bound to the GCN constructs at once. For example, if only two-thirds of the

binding sites that are occupied with Fl-C35 are displaced with the unlabeled C35, one

can still observe a significantly high polarization value compared to completely

unbound Fl-C35, which represents 100% inhibition.

Currently, we are examining various co-crystallization conditions of selected

peptides with both of the GCN constructs, which should provide an insight for

understanding the working mechanism of these short peptides.

Preliminary data for antiviral activities of selected peptides

The antiviral activity of P1-5 and P1-8 was investigated by collaborating with

Dr. Ron Geller in Prof. Judith Frydman’s lab at Stanford University. Briefly, hRSV

A2 strain was pre-mixed with 50 µM of each peptide and then added to Hep2 cells to

infect the cells. P5-7 and C35 peptides were included as negative and positive controls,

respectively. P5-7 (RCCHPNVPEISA) was initially identified from the panning

process; however, it has no measurable binding affinity to the 5HB as judged by the

FP assay. After 48 hours of infection, the media containing viral particles was diluted

and transferred to cells that were previously cultured in a 96-well plate. After 5 – 6


147

days of incubation, each plate was observed by a microscope for any signs of syncytial

formation in the wells. Preliminary data suggest that P1-5 and P1-8 showed some

inhibition in syncytial formation as an indication of antiviral activity (Fig. 5-11).

Although large batch-to-batch variations made it difficult to draw definitive

conclusions, it was obvious that these peptides lost some of their biological activity in

the cellular antiviral activity assay.

Figure 5-11. Peptides (P1-5 and P1-8) inhibition of hRSV infection in Hep2 cells 50 µM of

P1-5 and P1-8 were tested. 50 µM of P5-7 and C35 were included as negative and positive

controls, respectively.

Peptomeric hRSV inhibitors and their binding activity toward the5HB

The low antiviral activities of P1-5 and P1-8 in the cellular assays might be

caused by their relatively short length and consequently rapid proteolytic degradation

during the long course of the assays. This led us to explore the possibility of designing

peptomers based on selected 12-mer peptides by introducing peptoid residues to the

peptides sequences. As discussed earlier (Chapter 4), proline is the only naturally

occurring N-substituted cyclic amino acid, lacking a backbone NH group. For this


148

reason, proline has been recognized as a representative of peptoids in several

studies.44-48

To examine the effect of peptoid residue substitution at the proline residue

on the binding activity, we first synthesized analogues of P1-8 in which one proline

residue is located close to its C-terminus instead of testing P1-5, which contains two

proline residues.

As a starting point, NMEG and

NPhe were selected (Fig. 5-12). NMEG is

known to be very hydrophilic, which was

discussed in depth in Chapter 3 for its

potential applications in protein

modification, whereas NPhe is expected

to maintain a pattern of the P1-8

sequence, which contains alternating aromatic and charged residues. These two

peptomers, P1-8-NPhe and P1-8-NMEG, were manually synthesized and then purified

to > 90% purity (Fig. 5-13). Using the 5HB-based competitive FP assay, the binding

activities of these two peptomers at the concentration of 75 µM were determined (Fig.

5-14). Even though the concentration used in this study was lower than the IC50 value

of P1-8 (138 µM), we expected both P1-8 mimics to bind to the 5HB with similar

binding affinity based on our observation (Fig. 5-6). However, both P1-8-NPhe and

P1-8-NMEG significantly lost binding affinity to the 5HB, indicating that even single

peptoid substitution can affect the binding affinity of the peptide greatly. It might also

suggest that the determination of peptoid replacement needs more sophisticated

methods. For example, since P1-5 and P1-8 are unlikely to be helical, they may be

(A) (B)

N

O

O

N

O

Nmeg NPhe

Figure 5-12. Structures of peptoid residues to replace proline in P1-8 peptide (NMEG = N-methoxyethylglycine, NPhe = N-benzylglycine)


149

more amenable to the systematic screening approach described in Chapter 4. Thus it

may still be useful to investigate the potential of P1-8 (and possibly P1-5) as a design

scaffold for creating peptidomimetic antivirals.

H2N NH

HN

NH

HN

NH

HN

NH

HN

NH

N

O

O

O

O

O

O

O

O

O

O

NH

NH2

O

OOH

NHN

NH2

OH

O

NH2

HO

NH2O

NH

NH2O

NH

NH2HN

H2N NH

HN

NH

HN

NH

HN

NH

HN

NH

N

O

O

O

O

O

O

O

O

O

O

NH

NH2

O

OOH

NHN

NH2

OH

O

NH2

HO

NH2O

NH

NH2O

NH

NH2HN

O

(A)

(B)

Figure 5-13. Structures of P1-8 mimics Proline in P1-8 peptide was replaced with peptoid residues of NPhe and NMEG, yielding (A) P1-8-Nphe and (B) P1-8-NMEG, respectively.

Figure 5-14. Resulting data of P1-8 mimics using a competitive FP assay The binding of Fl-C35 to the 5HB was monitored in the presence and absence of 75 µM of each peptomer.


150


In this study we identified two promising 12-mer peptides, P1-5 and P1-8,

from a phage-displayed peptide library using our previously reported 5HB-based FP

assays. Even with their relatively short length, these peptides showed moderate

binding affinity to the 5HB. We also demonstrated that these dodecameric peptides

may inhibit viral replication by preventing syncytium formation in a cell-based assay,

however, their antiviral activity as hRSV fusion inhibitors needs to be further

confirmed.

Efforts to crystallize a complex of each 12-mer peptide with the GCN

constructs are currently being made. This will lead us to better understanding of the

binding mode of these peptides with the 5HB and enable maximizing their

performance as potential anti-hRSV agents.

Additionally, the binding of the peptomeric P1-8 mimics to the 5HB was tested

to prove the hypothesis that the proline residues can be replaced with peptoid residue.

Our rather disappointing results from a limited set of derivatives suggests that

transforming peptides to peptoids or peptomers will require more comprehensive

evaluation on each peptide residue depending on the system and furthermore, in-depth

side-chain optimization should be performed.


151


Materials for phage selection (Panning)

Ph.D.!-12 phage display peptide library was purchased from New England Biolabs

(Ipswich, MA, USA). The reagents for phage selection are as following: blocking

buffer (0.1 M NaHCO3, 5 mg/mL BSA, 0.02% NaN3 at pH 8.6), elution buffer (0.2 M

Glycine-HCl, 1 mg/mL BSA at pH 2.2), neutralization buffer (1 M Tris-HCl at pH

9.1), agarose top (10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 1g MgCl2, 7 g

agarose in 1 L water, the mixture is autoclaved, and then aliquoted into 50 mL tubes.

Solidified agarose top should be stored at room temperature, and melted in a

microwave prior to use.), LB/IPTG/Xgal plates (1 L LB medium, 15 g/L agar, 5 mg

IPTG in DMF, 4 mg Xgal [5-bromo-4-chloro-3-indoly-#-D-galatoside], the mixture is

autoclaved and then poured into the plate. The plate should be stored at 4 °C in the

dark), LB-Tet plates (1 L LB medium, 15 g/L agar, 20 mg tetracycline, the plate

should be stored at 4 °C.), PEG/NaCl solution (20% (w/v) polyethylene glycol-8000,

2.5 M NaCl), iodide buffer (10 mM Tris-HCl, 1.0 mM EDTA, 4 M NaI at pH 8.0),

TBS (50 mM Tris-HCl, 150 mM NaCl at pH 8.6), TBS-T (TBS with Tween-20).

Phage selection

Ph.D.!-12 phage display peptide library consists of ~ 2.7 x 109 electroporated

sequences, amplified once to yield ~55 copies of each sequence in 10 µL of the

supplied phage. Peptides are fused to the N-terminus of the protein of gene III, with a

GGS spacer and expressed on the phage surface in five identical copies. For phage

selection, wells in the 96-well plate were coated with 150 µL of 40 µg/mL 5HB at 4


152

°C overnight. Microplates were then blocked with 200 µL of blocking solution for 1 hr

at 4 °C. The plate was then washed with TBS-T (0.1%) and incubated with the phage

suspension for 1 hr at room temperature. Unbound phages were removed by washing

with TBS-T containing 0.1% (1st panning) and 0.5% (2

nd and 3

rd panning) of Tween-

20. Bound phage were eluted with 100 µL of 0.2 M glycine (pH 2.2) and neutralized

with 15 µL of 1 M Tris (pH 9.1). After amplification, DNA of eluted phage was

extracted and purified. DAN sequencing for each phage clone was done by Sequetech

(Mountain View, CA USA) using a -96 gIII primer (5’-CCCTCATAGTTAGCGT

AACG-3’, NEB).

Peptide synthesis

Peptide synthesis reagents were purchased from Applied Biosystems (Foster city, CA,

USA) or Sigma-Aldrich (Milwaukee, WI, USA). Fluorescently labeled peptide (Fl-

C35) with 95% purity was commercially obtained (EZBiolab, Carmel, IN, USA) and

used without further purification. Individual 12-mer peptides identified from the

panning were synthetically prepared with 95% purity (Bio Basic, Markham, Ontario,

Canada) and used without purification.

GCN constructs design, expression and purification

Synthetic genes for HRA-GCN (HG), and GCN-HRA-GCN (GHG) were

commercially prepared by Genescript (Piscataway, NJ, USA). Genes were

subsequently cloned into pET-15b using NdeI-BamHI restriction enzyme sites and the

resulting plasmids were transformed into Rosetta gami 2 (DE3). The resulting protein


153

sequences are following: for the HG construct, AVSKVLHLEGEVNKIKSALLSTN

KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKIKQIEDKIEEILSKIYHIEEI

ARIKKLIGEAGSGENLYFQGGSGGHHHHHHHH, and for the GHG construct,

ARIKQIEDKIEEILSKIYHIENEIARIKKLAVSKVLHLEGEVNKIKSALLSTNKA

VVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKIKQIEDKIEEILSKIYHIENEIA

RIKKLIGEAGSGENLYFQGGSGGHHHHHHHH (HRA domain in bold, GCN

domain in underlined, Tabacco Etch Virus (TEV) cleavage site in underlined bold and

His tag in underlined italic). Proteins were bacterially expressed with 1 mM ITPG for 3

hrs at 37 °C. The cells were harvested by centrifugation at 4500 g for 15 minutes, and

the cell pellet was resuspended in 20 mM phosphate buffered saline (PBS) and stored

at -80 °C until use. Cells were lysed with lysis buffer (CelLytic!B cell lysis reagent

[Sigma Aldrich, Milwaukee, WI, USA], 20 mM PBS at pH 7.4, 1 mM PMSF, protease

inhibitor cocktail [Sigma Aldrich, Milwaukee, WI, USA], 1% Triton X-100, 500 mM

NaCl and 0.2 mg/ml lysozyme) and incubated for 1 hr at room temperature. Cell lysate

was then clarified by centrifugation at 18,000 g for 30 min. The soluble fraction was

immediately incubated with a Nickel-immobilized chelating sepharose fast flow resin

(GE Healthcare, Piscataway, NJ, USA) at room temperature for 30 min with a gentle

agitation. The protein-bound resin was washed out with more than 10 column volumes

(CV) of a wash buffer (20 mM PBS at pH 7.4, 10 mM imidazole, 1% Triton X-100 and

500 mM NaCl). The GCN constructs were eluted with an elution buffer (20 mM PBS

at pH 7.4, 150 mM imidazole and 500 mM NaCl). The purity of the protein was

assessed by SDS-PAGE gel and the protein was used without further purification.


154

Phage binding assays using ELISA experiments

Binding studies were performed using ELISA to evaluate the specificity of binding of

selected 12-mer peptides to the 5HB. A microtiter plate was coated with 100 uL of

100 ug/mL of the 5HB in 0.1 M NaHCO3 (pH 8.6) and incubated at 4 °C overnight.

After rinsing wells with TBS-T (0.1%) and blocking with 5% milk in TBS-T (0.1%)

for 1 hr at 4 ºC, serially diluted phages ranging from 1012

to 108 phages/mL were

applied, followed by incubation for 2 hrs at room temperature with gentle agitation.

Unbound phage were removed by washing the plates 6 times with TBS-T (0.5%). 200

µL of horseradish peroxidase (HRP) conjugated anti-M13 antibody (Sigma Aldrich)

diluted in blocking buffer (1:5,000) was added and incubated at room temperature for

1 hr to detect phage interaction with the 5HB. After the addition of HRP substrate

(3,3’, 5,5’-tetramethylbenzidine, TMB) was added to each well, absorbance at 405 –

415 nm was measured. BSA was used as a negative control.

Competitive FP binding assay

Competitive FP binding assays of selected 12-mer peptides were carried out as

previously reported.30

Briefly, using a 5HB protein construct and a fluorescently

labeled 35 amino acid-long peptide (Fl-C35) as a target and a tracer respectively, the

binding affinities of dodecameric peptides were individually evaluated. Each well in a

black 96-well plate (Corning Inc. Lowell, MA, USA) contained 20 nM of the 5HB and

increasing concentrations of each 12-mer peptide in FP buffer (20 mM PBS at pH 7.4,

500 mM NaCl, 0.01% (v/v) Tween-20, and 0.05 mg/ml bovine gamma globulin) in a

final volume of 185 µL with 1 hr incubation at room temperature. Fl-C35 was then


155

added to a final concentration of 5 nM followed by 30 min incubation at room

temperature. The FP responses were monitored using a Synergy4 (Biotek, Winooski,

VT, USA) plate reader with an excitation wavelength of 485 nm and an emission

wavelength of 530 nm. The percentage of inhibition (% Inhibition) was calculated

using the following equation: % Inhibition = 100$[(mP-mPf)/(mPb-mPf)], where

unbound Fl-C35 (mPf), bound Fl-C35 (mPb) and the bound inhibitor to the 5HB (mP)

are used accordingly.



MD, USA). Samples were prepared in 20 mM PBS, pH 7.4 in concentration of 50 µM.

Data were recorded from 195 to 260 nm with a scanning speed of 20 nm/min and a

bandwidth at 1.0 nm in a 0.1 cm path-length quartz cell. Each CD spectrum was an

average of 3 measurements and corrected for a buffer blank obtained under identical

conditions. The resulting data was converted to per-residue molar ellipticity units, [!]

(deg%cm2%dmol

-1%residue

-1).

Peptomer synthesis

Each peptomer (P1-8-NPhe and P1-8-NMEG) were manually synthesized in the

laboratory using standard Fmoc chemistry for peptide residues and a submonomer

peptoid synthesis49

for peptoid monomers manually on rink amide resin. After

synthesis, the peptides were cleaved off the resin and deprotected in TFA/water/

TIPS/thionisole (90:5:2.5:2.5 v/v) for 1.5 hr at room temperature with agitation.


156

Peptomers were purified by preparative RP-HPLC on a C18 column using a linear

gradient of 5-99% solvent B in solvent A over 60 min (solvent A is 0.1% (v/v) TFA in

water and solvent B is 0.1% (v/v) TFA in acetonitrile). Final purities of synthetic

peptomers were confirmed to be > 90% by analytical RP-HPLC, and the molecular

weight of the purified product was confirmed by electrospray mass spectrometry (ESI)

at the Stanford University Mass Spectrometry (SUMS) facility.


157

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163

Chapter Six

Conclusion


In this thesis, we have validated a protein-based HTS-compatible fluorescence

polarization (FP) assay as a successful screening platform for hRSV fusion inhibitors,

and evaluated the potential of short peptides and their peptoid-based peptidomimetics

using this assay. To obtain therapeutically favorable properties including improved

solubility and enhanced half-lives, we also have demonstrated a broadly-applicable

methodology for creating peptoid-based biostable therapeutic peptidomimetics.

In Chapter 2, we discussed that the 5HB-based FP assay might also provide a

key to study small molecule antivirals whose mechanism of action is still poorly

understood. Our recent findings in testing several small molecule fusion inhibitors

using the 5HB-based FP assay suggest that small molecules might not block the 6HB

formation of hRSV F as others have claimed,1-5 but likely trap the 6HB in a non-

functional form.6 Because of the relatively small size of the antivirals, it is likely that

the bulk of the HRB helices can still interact with the HRA helices to form a partially

functional 6HB in the presence of the bound small molecule, while the remaining part

of the HRB helices covered over the small molecules bound to the hydrophobic

pocket. Simply by varying the length of the probe peptide or the region where the

probe is binding (e.g., covering the hydrophobic pocket or not), it might be possible to

narrow down the possible binding mode of the small molecules. However, to prove the

Chapter Six: Conclusions and future prospects

164

hypothesized mechanism of the small molecules using the 5HB, the most important

question to answer is whether or not these small molecules bind to the hydrophobic

pocket (i.e., the known binding site for Fl-C35) in the 5HB. One simple test can be to

monitor the small molecule binding to the 5HB in the presence and absence of C35 or

shorter derivatives by thin layer chromatography (TLC), which has been widely used

for the separation and identification of biochemical compounds such as carbohydrates,

steroid hormones and amino acids.7 After incubation, the unbound small molecules

can be removed from the mixture by a desalting column and only the 5HB-bound

small molecule-bound 5HB will be recovered. On a TLC plate, small molecules will

be separated from the sample spot at the baseline and will migrate with the mobile

phase (e.g., dichloromethane), allowing then to be detected.

Subsequently, the structural studies of the small molecule inhibitors with the

whole hRSV F protein should also be pursued to understand the interaction of small

molecules with the hRSV F protein in depth. Electron microscopy (EM) studies might

be a simple experiment to begin with, since EM studies of the F proteins from other

paramyxoviruses have shown that the pre- and post-fusion structures of the F protein

can be easily distinguished and the sample preparation would be relatively

straightforward compared to other methods (e.g., NMR studies).8 In the presence and

absence of the small molecules, the hRSV F protein is expected to behave differently

upon activation to the post-fusion form. To gain further insight on the interaction

between the small molecules and the hRSV F protein at the atomic level, X-ray

structural studies of a complex of the small molecules with the full length hRSV F

should be pursued as a long term strategy.


165

In Chapter 3, we suggest that NMEGylation can be applied to modify a

simple, short therapeutic peptide as well as proteins yielding highly monodisperse

products with saved time and costs. NMEGylated peptide/peptoid libraries could be

designed at an early stage of molecular optimization varying both the number and

position of Nmegs and any spacers due to the intrinsic compatibility of NMEGylation

with solid phase peptide/peptoid synthesis.

Our proposal in this study can be further confirmed if we can prove how

having a linker helps to retain the binding affinity of the C20 peptide. To fully

understand how placing a glycine linker in between NMEG and the C20 peptide

sequence affects the binding affinity, structural studies using X-ray crystallography

might provide useful information. We generated a possible 3-D model of NMEG-Gly-

C20 binding to the 5HB using Pymol.9 The model reveals a possible H-bond between

the hydrogen atom at N-terminus (donor) and the nitrogen atom at Lys191 (acceptor)

from one of the HRA helices, showing a distance of 2.7 Å (Fig. 6-1). This postulated

H-bond enabled by the glycine linker might be very weak since it occurs between

primary and secondary amine, however, it might act as a stabilizing factor to place

Isoleucine (the first residue of the C20) in the right position to easily initiate binding of

the C20 to the 5HB, which may explain why NMEG-Gly-C20 could recover most of the

binding affinity to the 5HB compared to the other linker-less NMEGylated peptides.


166

Figure 6-1. Stabilized the binding of NMEG-Gly-C20 to the 5HB by H-bond Three HRA helices are shown in green ribbons and the C20 peptide is presented as white ribbon. The first residue of the C20 peptide, Isoleucine, is represented by red, and Lysine at position 191 in the HRA helix is shown in magenta. The possible hydrogen bond between NMEG residue and Lys191 are shown in dotted line.

Additionally, we reasoned that since a large open contact surface is involved in

the binding of the C20 to the 5HB, direct conjugation of oligoNMEG led to impeded

binding of NMEGylated peptides to the 5HB due to possible steric hindrance. As

illustrated in Fig. 6-2, we believe NMEGylation will more successfully improve the

biophysical properties of ligands that involve a narrow binding cleft (e.g., substrate

and enzyme) without severely impacting their biological activity. Such a finding

would further demonstrate the potential of NMEGylation as a biotherapeutic

modification tool.


167

Figure 6-2. Retaining biological activity of NMEGylated ligands and steric hindrance (A) When the binding site is narrow and limited to several active residues, oligoNMEG in red can be directly attached to a target ligand (e.g., the C20 peptide and substrates) without deteriorating the biological activity of ligands. (B) The high steric hindrance in dotted circle may cause the lower binding affinity of NMEGylated ligands that interact a large binding interface on a receptor (e.g., the 5HB and enzymes)

The combined peptide scan approach using alanine, proline, and sarcosine for

determining peptoid-replaceable peptide residues in therapeutic peptides that we

proposed in Chapter 4 can be applied to design peptidomimetic hRSV fusion

inhibitors using the dodecameric peptides identified in Chapter 5 as a design basis.

Two promising 12-mer peptides, P1-5 and P1-8, showed a moderate binding affinity

(27 and 138 µM respectively) to the 5HB, which is comparable with the C20 peptide.

To improve the likely high susceptibility to proteases caused by their short length,

simple P1-8 analogues (Pro ! peptoids) were tested, however, in part because of the

small number of peptomers, the results were rather disappointing. Therefore,

alanine/proline/sarcosine substitution studies will provide critical information on each

residue to design better peptoid-based peptidomimetic peptides based on the 12-mer

peptides.

As discussed in Chapter 5, the avidity effect caused by multivalency of phage-

bound peptides has been proposes to explain the lower biological activity of


168

individually synthesized peptide with the target proteins. However, if we can mimic

the avidity effect by intentionally multimerizing peptides, it may be possible to gain

positive and desirable impact on the biological activity.

First, dimerization via a disulfide bond10 and native chemical ligation

methods11,12 have been widely used to provide increased structural stability and thus

enhance biological activity. However, since none of 12-mer peptides contain cysteine,

it should be critical to find optimal sites for cysteine mutation or insertion. The

stabilizing effect of the disulfide bond in peptides or proteins can be varied depending

on where the disulfide bond introduced.13 Once cysteine-contained peptides are

prepared, the disulfide bonds are formed simply by air oxidation of a solution of

reduced peptides.

Second, trimeric peptides can be prepared using a scaffold that provides three

functional groups to tether the target peptide. This strategy has been applied to

peptides derived from HIV-1 gp41 HRA and HRB helices, generating 3-helix bundle

mimetics of HIV-1 gp41. (Fig. 6-3).14 Trimeric 12-mer peptides can be generated by

using a trimeric scaffold such as TBB (Tris-(bromomethyl) benzene) or TREN (Tris-

(2-aminoethyl)amine) (Fig. 6-4). TREN has been used as an effective structural

scaffold for the assembly of triple helical collagen mimetic structures.15 As compared

to TBB, TREN is expected to provide a flexible tripodal structure, which allows for a

better accommodation of the three-peptide chains.


169

O

HN

HN

NHO O

O

O

NH

HN

O

OO

NHO

O

!Ala - Lys2 - Lys1 -N

SCO

OC

N

SCO

OC

N

SCO

OC

Gly

S T

Cys-peptide

(A) (B)

Lys1

GlyCO

Lys2

CO

CO

!Ala

Figure 6.3 Schematic diagram of (A) a scaffold for trimeric HIV-1 gp41 mimetics and (B) the resulting trimeric helix bundle (S = space sequence, T = T-helper sequence for vaccine purpose) Authors claimed that this scaffold could bear a unique dual function as an antiviral agent as well as a synthetic vaccine (or other peptides of interest). Images are adapted from Tam et al. (2002) Org. Lett. 4, 23, 4167.

(A) (B)

Br

Br

BrN

NH2

H2N NH2

(A) TBB (B) TREN

Figure 6.4 Structures of (A) Tris-(bromomethyl) benzene (TBB) and (B) Tris-(2-aminoethyl)amine (TREN) TREN has been used an effective structural scaffold for the assembly of triple helical collagen mimetic structures, allowing for a better accommodation of the three peptide chains with a flexible tripodal structure.

Third, selected 12-mer peptides can even further multimerized using a peptide

dendrimer approach (more than 3 branches), which has been recognized for potential

therapeutic applications including drug delivery,16 multiple antigenic peptides,17

antiviral peptides18,19 and antimicrobial peptides.20 Since dendrimers have highly

branched macromolecules synthesized from a highly structured core (Fig. 6.5), they

can present multiple ligands or binding sites, which mimics the avidity effects.17,21


170

It is anticipated that multimerized peptides using the strategies described above

will present high protease resistance and increased local concentration, thereby having

longer half-lives and tighter binding to the target protein.

(A) (B)

NN

N

N

N

N

NH2

NH2

NH2H2N

N

N

NH2

NH2

NH2

NH2

N

N

N

NH2

H2N

H2N

H2N

N

N

N

NH2H2N

NH2

H2N

H2N

HN

NH

HN

HN

NH

NH2

O

O

HN

NH2

O

H2N

HN

OHN O

HOOC

O

O

NH

NH

O

HN

OH2N

NH2

OH2N

NH2

NHO

HN

O NH2

H2N

NH

NH2

ONH2

H2N

O

HN

O

NH2

NH2

Figure 6.5 Schematic representations of peptide dendrimer motif examples (A) poly-(prolylene imine) core is currently commercially available and (B) lysine-based core

In summary, we describe a novel and effective high-throughput screening

compatible platform for screening potential viral fusion inhibitors (Chapter 2). We

used both knowledge-based (Chapter 2) and phage-displayed peptide library

(Chapter 5) approaches to generate possible ”hits” for such inhibitors. We then report

two completely novel and broadly-applicable approaches for optimizing therapeutic

candidate peptides into peptomers. We report NMEGylation as a method to “decorate”

peptides for improved solubility and protease resistance (Chapter 3) and a

comprehensive Ala/Pro/Sar scan method for identifying optimal residues in a peptide

for a peptoid substitution. The work has greatly focused on creating enabling tools,

such as the 5HB screening platform and the peptomer optimization strategies, and thus

expect that this work will have a wide and lasting influence.


171

References

1. Bonfanti, J.F. & Roymans, D. Prospects for the development of fusion inhibitors to treat human respiratory syncytial virus infection. Curr Opin Drug

Discov Devel 12, 479-487 (2009).

2. Cianci, C., Langley, D.R., Dischino, D.D., Sun, Y., Yu, K.L., Stanley, A., Roach, J., Li, Z., Dalterio, R., Colonno, R., Meanwell, N.A. & Krystal, M. Targeting a binding pocket within the trimer-of-hairpins: small-molecule inhibition of viral fusion. Proc Natl Acad Sci U S A 101, 15046-15051 (2004).

3. Douglas, J.L., Panis, M.L., Ho, E., Lin, K.Y., Krawczyk, S.H., Grant, D.M., Cai, R., Swaminathan, S. & Cihlar, T. Inhibition of respiratory syncytial virus fusion by the small molecule VP-14637 via specific interactions with F protein. J Virol 77, 5054-5064 (2003).

4. Razinkov, V., Huntley, C., Ellestad, G. & Krishnamurthy, G. RSV entry inhibitors block F-protein mediated fusion with model membranes. Antiviral

Res 55, 189-200 (2002).

5. Andries, K., Moeremans, M., Gevers, T., Willebrords, R., Sommen, C., Lacrampe, J., Janssens, F. & Wyde, P.R. Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus. Antiviral Res 60, 209-219 (2003).

6. Roymans, D., De Bondt, H.L., Arnoult, E., Geluykens, P., Gevers, T., Van Ginderen, M., Verheyen, N., Kim, H., Willebrords, R., Bonfanti, J.F., Bruinzeel, W., Cummings, M.D., van Vlijmen, H. & Andries, K. Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein. Proc Natl

Acad Sci U S A 107, 308-313 (2010).

7. Bhushan, R. & Martens, J. Peptides and Proteins. in Handbook of Thin-Layer

Chromatography (eds. Sherma, J. & Fried, B.) (CRC press, 2003).

8. Connolly, S.A., Leser, G.P., Yin, H.S., Jardetzky, T.S. & Lamb, R.A. Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy. Proc

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9. DeLano, W.L. The PyMOL Molecular Graphics System. (DeLano Scientific LLC., Palo Alto, CA, USA.).

10. Dempsey, C.E., Ueno, S. & Avison, M.B. Enhanced membrane permeabilization and antibacterial activity of a disulfide-dimerized magainin analogue. Biochemistry 42, 402-409 (2003).

11. Xiao, J., Hamilton, B.S. & Tolbert, T.J. Synthesis of N-terminally linked protein and peptide dimers by native chemical ligation. Bioconjug Chem 21, 1943-1947 (2010).

12. Xiao, J. & Tolbert, T.J. Synthesis of N-terminally linked protein dimers and trimers by a combined native chemical ligation-CuAAC click chemistry strategy. Org Lett 11, 4144-4147 (2009).

13. Zhou, N.E., Kay, C.M. & Hodges, R.S. Disulfide bond contribution to protein stability: positional effects of substitution in the hydrophobic core of the two-stranded alpha-helical coiled-coil. Biochemistry 32, 3178-3187 (1993).

14. Tam, J.P. & Yu, Q. A facile ligation approach to prepare three-helix bundles of HIV fusion-state protein mimetics. Org Lett 4, 4167-4170 (2002).

15. Kwak, J., De Capua, A., Locardi, E. & Goodman, M. TREN (Tris(2-aminoethyl)amine): an effective scaffold for the assembly of triple helical collagen mimetic structures. J Am Chem Soc 124, 14085-14091 (2002).

16. Wolinsky, J.B. & Grinstaff, M.W. Therapeutic and diagnostic applications of dendrimers for cancer treatment. Adv Drug Deliv Rev 60, 1037-1055 (2008).

17. Niederhafner, P., Sebestik, J. & Jezek, J. Peptide dendrimers. J Pept Sci 11, 757-788 (2005).

18. Luganini, A., Giuliani, A., Pirri, G., Pizzuto, L., Landolfo, S. & Gribaudo, G. Peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate. Antiviral Res 85, 532-540 (2010).


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