Peptide in Diagnostik und Therapie

Annette G. Beck-SickingerfüInstitut für Biochemie

Universität Leipzig, Deutschland

Peptides Drugs ???Peptides …. Drugs ???

Major problems

• Difficult to synthesize in large quantities• Low stability• Low bioavailability• Low bioavailability• No oral application

Harris, J. M. et. al, Nature Reviews Drug Discovery, 2003, 2, 214 – 221.

M j blMajor problems

• Difficult to synthesize in large quantities• Low stability• Low bioavailabilityFuzeon (T20, 36mer peptide)

peptide blockbuster to target HIV• Low bioavailability• No oral application

peptide blockbuster to target HIVHIV entry inhibitor

CH3CO-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-NH2

gp41

PTM TM CPFP Heptad repeat 1(HR1) Heptad repeat 2(HR2)Loop530 593 620 663

Loop PTM TM

675638T20

C’NPTM TM CPFP Heptad repeat 1(HR1) Heptad repeat 2(HR2)Loop

546 581 628 661

Loop PTM TM

N36 C34

X-ray‘Hairpin’ intermediate

Chan and Kim (1998)

M j blMajor problems

• Low stability, low bioavailability

Exenentide (GLP1 agonist)( g )Liraglutide – lipidatedIcatibant – Bradikinin antagonist( t i i i id )(non proteinogenic amino acids)EPO mimetic - pegylated Icatibant

Liraglutide

M j blMajor problems

• No oral application

Subcutan: Degarelin, Abarelix (GnRH Antagonist); Icatibant (Bradikinin Antagonist)

SC i l t V tid (SST A i t)SC implant: Vapreotide (SST-Agonist)

Liposomal BLP-25 (mucin inhibitor)Liposomal BLP 25 (mucin inhibitor)

Inhalation: Aviptadil (VIP Agonist)

Proteins & Peptides – New Hopes in Drug Development...

• USA 2009: • Covering 150 diseases• HIV/AIDS

• Market – 80 polypeptide drugs

• About 1/3 of all drugs

• HIV/AIDS• Cancer• Autoimmune diseases

Di b t• About 1/3 of all drugs in clinical trials are polypeptides

• Diabetes

L t i d di t bl i th i i i b h iLess toxic and more predictable in their in vivo behaviorLarge interaction sitesNot immunogenic in most casesMetabolic products non toxicMetabolic products non toxic

Frokjaer, S. et. al, Nature Reviews Drug Discovery, 2005, 4, 298 – 306.Harris, J. M. et. al, Nature Reviews Drug Discovery, 2003, 2, 214 – 221.

Peptide Pharmaceuticals in Clinical Phase III Targeting GPCRs

Drug Indication Action

Aviptadil SarcoidosisPulmonary hypertensionAutoimmune disease

Agonist of vasoactive intestinal peptide receptors

Autoimmune diseaseRespiratory distress syndrom

Salmon calcitonin (oral) Osteoarthritis Agonist of calcitonin receptorSalmon calcitonin (oral) OsteoarthritisOsteoporosis

Agonist of calcitonin receptor

Tedoglutide Gastrointestinal diseaseInflammatory bowel

Glucagon-like peptide 2analogueInflammatory bowel

diseaseCrohn's disease

analogue

Teriparatide Osteoporosis Agonist of parathyroid p p g p yhormone receptor

Terlipressin Hepatorenal syndromeRenal failure

Agonist of vasopressinreceptor

Tesamorelin Bone injurySleep disorderCystic fibrosis

Agonist of growth hormone releasing hormone receptor

Neuropeptide Y Family

C t t iti

NPY PPPYYConstant positionsamong all species 2222 1515 77

NPY

5 12 27 353398

Y P S KP DNP GE DAP AE DMARY Y S AL RHY I NL I T RQRY

PP

PYY Y P I KP E AP GE DAS P E E L NRY Y AS L RHY L NL V T RQRY

AP L E P V Y P GDNAT P E QMAQY AADL RRY I NML T RP RYPP AP L E P V Y P GDNAT P E QMAQY AADL RRY I NML T RP RY

Neuropeptide Y: NPY; Peptide YY: PYY; Pancreatic Polypeptide: PP

Neuropeptide YNeuropeptide Y Y4/PP1-Receptor:3Y4/PP1-Receptor:3Neuropeptide YNeuropeptide Y PP, cloned, 375 aacAMP inhibitionPP, cloned, 375 aacAMP inhibition

food intake

Y RY R

Y5-Receptor:cloned, 455 aaG Protein coupled

Y5-Receptor:cloned, 455 aaG Protein coupledY1-Receptor:

cloned, 384 aaG-Protein coupledCa2+ mobilization

Y1-Receptor:cloned, 384 aaG-Protein coupledCa2+ mobilization

Y2-Receptor:cloned, 381 aaG-Protein coupled

Y2-Receptor:cloned, 381 aaG-Protein coupled food intake

G-Protein coupledcAMP inhibitionG-Protein coupledcAMP inhibition

NH2

Ca mobilizationcAMP inhibitionCa mobilizationcAMP inhibition

G Protein coupledcAMP inhibitionCa2+-channels

G Protein coupledcAMP inhibitionCa2+-channels

vasoconstriction,food intakecancer

noradrenaline release inhibition of GABAergic neurons

epilepsy memory retensioncancer epilepsy, memory retensionCOOH

adenylate cyclase

cAMPcAMP

Approches to drugable Y-ligands

• Stabilize hPP for application in t b li dimetabolic disease

• PP: satiety hormone of the gut!• Agonists to induce satiety• How can we modify hPP?y

• Y receptor selective analogues in• Y1-receptor selective analogues in tumor targeting

Glucagon

PP

Orexin

Schwartz MW, Morton GJ. Nature 2002;418:595-7

Cycle MutagenesisCycle Mutagenesis

hPP/NPY

Y R Y RY R Y R

NPY

NH2R33

R25Y1R, Y4RNH2Y2R, Y5R

R25D5.27

NH2

R35R33R35 D6.59 D6.59

R25D5.27

TM5 TM7CONH2

TM5 TM7CONH2

D

TM5

TM6

TM7TM5

TM6

TM7

N. Merten et al., J. Biol. Chem. 2007, 282, 7543

Further InteractionsFurther Interactions …pancreatic polypeptide neuropeptide Y

D. Lindner et al., Biochemistry 2008, 47, 5905

Ligand-Receptor Interaction …Ligand Receptor Interaction …

Model: Kristian Kaufmann, Jens Meiler, Vanderbilt University, TN

Characterisation of the hPP analoguesg

Peptide Activity at hY4 (nM)

Half life in h blood% intact peptide after 24 h

Structure by CD

Helicity (%)after 24 h Helicity (%)hPP 2.2 56 % 48

hPP(K4Pam) >100 80 % 46hPP(K4Pam) >100 80 % 46

hPP(K7Pam) 98 88 % 42

hPP(K16Pam) >100 95 % 44

hPP(K22Pam) 10.1 90 % 48

hPP(K30Pam) 21.1 91 % 43

hPP(K30PEG2000) 15 5 90 % 43hPP(K30PEG2000) 15.5 90 % 43

Pam: palmitoylated PEG: pegylated

Liver homogenatesLiver homogenates

• Unmodified hPP: 15 min half life• Pegylated hPP: 52 min half life• Lipidated hPP: 283 min half life• Lipidated hPP: 283 min half life

CF l b l d l i b ti tiCF-labeled analogue, incubation, separationMS/MS analysis: major cleavage sites

In vivo studies – 68Ga-LabelingIn vivo studies Ga Labeling

OO

ONH2

NNN

NHOOC

GaNH

O O

DOTA

DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

In vivo studiesIn vivo studies

Blood samples (400 µL) after 1, 3, 5, 10, 20, 30 and 60 min

Peptide

PET studies

Biodistribution Studies

↓

withdrawal of remaining urine and blood

removal and investigation of bladder, kidney, liver, lung, y, , g

Approches to drugable Y-ligands

• Slight loss in activity, no change in t tstructure

• Lipidisation and pegylation increases p p gystability in vitro and in vivo

• Organ specific targeting by modificationOrgan specific targeting by modification

A l i t t ti Y1• Analogues in tumor targeting Y1-receptor selective

Neuropeptide Y and TumorNeuropeptide Y and Tumor ...

tumor normal breast tissuetotal binding of 125I [L,P] NPY

[L,P] NPY

PYY3-36PYY3 36

total binding of 125I PYY total binding of 125I PYY 3-36g

Reubi et al., Cancer Res. 2001,61, 4636

Y2 Y1 change during neoplastic transformation

Binding of NPY at MCF7 Cells

3250

3500NPYm

]

(Y li d)

2750

3000

3250 Ala31,Aib32-NPYLeu31,Pro34-NPY

H-N

PY [d

p

mRNA

(Y5-ligand)(Y1,Y5-ligand)

2250

2500

2750

boun

d3 H mRNA

-11 -10 -9 -8 -7 -6 -52000

log [peptide]log [peptide]

Y receptors ept

or

PD

H

Y1 receptorsIC50: 1.3nM

Y1-

rece

GA

P

Diagnosis TherapyY receptor selectivePeptide

Diagnosis TherapyY1-receptor selective agonists:

[F7, P34] pNPY[ , ] p

99mTc, 67/68Ga111In, 18F

receptorY1-receptorreceptorY1 receptor

visualization treatment

Trafficking of GPCRsAgonist

Limbird et al., Molecular Interventions 4, 326-336 (2004)

Arrestin Mediated Internalisation of Y1-YFP Receptors

PreTransfection of stable HEK 293-hY1-EYFPHEK 293 hY1 EYFP cells

with 100ng ARR35 min

with 100ng ARR3-mCherry,

I b ti ith 1 M

20 min

Incubation with 1 µM NPY

20 min

Collaboration with Seva Gurevich, VU, Nashville30 min

Selective Agonists (Ag.) and Antagonists (Ant.)

HEK293-hY1R-EYFP cells; 60 min; 37°C

BHK-hY2R-EGFP cells; 60 min; 37°C

[F7 P34] NPY [Ahx5-24] NPY

hY2R

[F7 P34] NPY [Ahx5-24] NPY

hY1R EYFPEGFP

[F7, P34]-NPY [Ahx5 24]-NPY[F7, P34]-NPY [Ahx5-24]-NPYY1-Ag.

Y2-Ag.

Y1-Ag.

Y2-Ag.

PP [Ala31, Aib32]-NPYPP [Ala31, Aib32]-NPYY4- Y5- Y4- Y5-Y4Ag.

Y5Ag.

Y4Ag.

Y5Ag.

random NPYBIIE0246random NPYBIBP3226

Y1-Ant

Y2-Ant

10 µm 10 µm

Ant Ant

Internalization of TAMRA-NPYInternalization of TAMRA-NPY 10 min 1 µM TAMRA‐NPY then 1 µM NPY 

TAMRA-Peptide

Merge hY2_EGFPTAMRA‐NPY

Control 10 min 1 µM NPY

ReceptorReceptor

Internalisation of Receptors

HEK293-hY1-EYFP cells + hY2-ECFP: F7, P34 and Ahx(5-24)

t l [Ah (5 24)] NPY[F7 P34] NPYcontrol [Ahx(5-24)] NPY[F7, P34] NPY

atio

nst

imul

a

Y1-agonist Y2-agonist

YFP: yellow fluorescent protein, CFP: cyan fluorescent protein Böhme et al. Cell. Signal. 20 (2008) 1740

Principles in Labelling

[Phe7,Pro34] NPY

O

N NCO2HHO2C

111InO

NNNH

HO

H

N NCO2HHO2C

XCOOC

CH2-CO-

CO

NH-NPY

DOTA His(ac) 99DOTA His(ac) X=Re, 99mTc

D. Zwanziger et al. (2008) Bioconjug Chem. 19:1430

Labelling of NPY with Re(CO)3-(NαHis-ac)

O O NH NPY R iO

Labelling of NPY with Re(CO)3 (N His ac)

OBr DICO

O OBr Br

-H2O

NH2-NPY- Resin

NNH-His(Trt)-OtBu

Br NH-NPY- ResinOBr

2

OH

CH2N

O-O-C-CH-NH2

( )- HBr

CH2N

N

O-O-C-CH-NH

95% TFA / 5% Scavenger

H

CH2N

N

O

OHO-C-CH-NH

NH-NPY- ResinO

NH-NPY

ONH

HO

H

XCOOC

NNCH2-CO-

CO

pH 4.3Incubation 2 hours

5.0 % excess Re-SaltNH-NPY

X = 99mTc or 188Re

I. Khan, D. Zwanziger et al., Angew. Chem. (2010) 49:1155.

AgonismAgonism

1c Re(CO)3-(NαHis-ac)-NPY

1d Lys4(Re(CO)3 –(NαHis-ac))-NPY

2c Re(CO)3-(NαHis-ac) -[Phe7, Pro34]NPY2c Re(CO)3 (N His ac) [Phe , Pro ]NPY

2d Lys4(Re(CO)3 –(NαHis-ac)) -[Phe7, Pro34]NPY

InternalisationInternalisation

1c Re(CO)3-(NαHis-ac)-NPY

1d Lys4(Re(CO)3 –(NαHis-ac))-NPY

2c Re(CO)3-(NαHis-ac) -[Phe7, Pro34]NPY

2d Lys4(Re(CO)3 –(NαHis-ac)) -[Phe7, Pro34]NPY

Stability and Protein Binding inStability and Protein Binding in Human Blood

2c Re(CO)3-(NαHis-ac) -[Phe7, Pro34]NPY

2d Lys4(Re(CO)3 –(NαHis-ac)) -[Phe7, Pro34]NPY

2a 99mTc-(NαHis-ac) -[Phe7, Pro34]NPY

2b Lys4 (NαHis-ac- 99mTc) -[Phe7, Pro34]NPY

Healthy VolunteerHealthy Volunteer

no uptake in breast ofno uptake in breast of99mTc(core)3+-(NHis-ac)-[Phe7, Pro34]NPY

99mTc labelled NPY PeptideTc labelled NPY Peptide

99mTc-MDP99mTc(core)3+-(NHis-ac)-[Phe7, Pro34]NPYMDP: Methylendiphosphonate

Comparison of NPY-peptide and MDP-ScanComparison of NPY peptide and MDP Scan

I. Khan, D. Zwanziger et al., Angew. Chem. (2010) 49:1155.

Diagnosis TherapyPeptide

Diagnosis TherapyY1-receptor selective agonists: [F7, P34] pNPY

99mTc, 67/68Ga111In, 18F

188Re

receptorY1-receptorreceptorY1 receptor

visualization treatment

S mmarSummary

Peptides might be useful drugs: metabolic disease, tumor

Modification improve stability, targeting

Possibility of targeted tumor therapy and diagnosis

Thanks a lot! Krummenhennersdorf 2009Thanks a lot!- external retreat -

Dr. Diana LindnerDr. Ilka BöhmeDr. Irfan Khan

V ikCornelia Walther

Denise Zwanziger Verena

Ahrens

VeronikaMäde

Dr. KarinMörl

Ahrens

Dr. Jan StichelKathrin Bellmann-Sickert

Tom Marten KilianAnette SchreiberStefanie Nagel

Collaborators ….Neuropeptides/Receptors – Assays/Cells, TissuesProf. W. Colmers, EdmontonProf. H. Herzog, SydneyProf T W Schwartz Copenhagen

Clinical StudiesProf. J. Reubi, Univ. BernProf. W. Kiess, Univ. LeipzigProf M Stumvoll Univ LeipzigProf. T. W. Schwartz, Copenhagen

Prof. H. Cox, LondonProf. T. Schöneberg, Leipzig University Prof. S. Gurevich, VU, Nashville

Radiopharmacy

Prof. M. Stumvoll, Univ. LeipzigProf. Javed, Nuclear Medicine

and Oncology, Lahore

RadiopharmacyProf. August Schubiger, ETH Zürich

and PSI Würenlingen, CHDr. Bergmann, FZ Rossendorf

Molecular ModellingProf. J. Meiler, VU, Nashville

gDr. Lehmann, Dr. Stellfeld

Bayer-Schering Healthcare AGStructural Biology/Protein ExpressionProf. R. Rudolph, MLU HalleProf. D. Huster, Leipzig UniversityP f N St ät L i i U i it

ChemistryProf N Metzler Nolte Univ Bochum

$-€-SFrGerman Research Foundation (DFG)DAAD

Prof. N. Sträter, Leipzig University

Prof. N. Metzler-Nolte, Univ. BochumProf. E. Hey-Hawkins, Leipzig UniversityProf. M. Mutter, EPFL Lausanne

ESF, EFRESchweizer KrebsligaBayer-Schering Healthcare AG

Biochemistry/Univ. Leipzig Fond Chemischer Industrie