Activity-Based Protein Profiling

Contents

Introduction-Assignment of Protein Function In the Postgenomic Era -Detection Strategies for Activity-Based Proteomics-What Is an Activity-Based Probe

Application of Activity-Based Probes-Identification of Biomarkers for Human Disease -In Vivo Imaging of Enzyme Activities-Small Molecule Screening and Target Discovery

Case-discussion -p90 ribosomal protein S6 kinases-Metalloprotease -Cysteine protease

Conclusion

Global analysis of changes in gene transcription and translation by abundance-based

genomic and proteomic approaches provides only indirect information about protein

function.

* In the postgenomic era researchers are now confronted with the task of assigning

functions to tens of thousands of proteins.

Assignment of protein function in the postgenomic era

Many proteins, such as enzymes, are functionally regulated by a series of post-

translational mechanisms, leading to a lack of correlation between activity and

expression levels.

Activity-based protein profiling (ABPP) is a chemical strategy that utilizes active site

directed covalent probes to profile the functional state of enzymes in complex

proteomes.

Detection strategies for activity-based proteomics

* Unraveling the functional roles of proteins is a major challenge facing the

post-genome researcher

Advances towards this goal have been made through the development of

both chemical and biochemical tools for monitoring protein activity

1. Small-molecule substrate reporters of enzymatic activity

2. Protein-based reporters of enzymatic activity

3. Activity-based probe

• Examples (3 becomes more popular now)

Small-molecule substrate reporters of enzymatic activity

* These reagents carry fluorescent groups, and thus energy emission upon

their enzymatic conversion to product can be monitored over time

1.The majority of basic fluorogenic probes cannot be directly applied to complex cellular environments 2. The another challenge in using the approach lies in the ability to generate probes that are specific for an individual enzyme (a peptide has the potential to function as a substrate for more than one class of proteolytic enzymes)

TRENDS in Cell Biology, Vol.14 No.1 January 2004

Disadvantages:

Protein-based reporters of enzymatic activity

•Fluorescent reporters •Bioluminescent reporters

1.The use of FRET has been extended further to design biochemical tools for

monitoring enzymatic activity inside cells 2. They all suffer from the selectivity of probes for a specific enzyme target

TRENDS in Cell Biology, Vol.14 No.1 January 2004

Disadvantages:

What Is an Activity-Based probe (ABP) ?

3. A tag, which is used to visualize the modified enzyme

* The activity-based probes (ABPs): they generally contain three main

functional groups:

2. A linker region, which can be specific for different enzymes

1. The chemical reactive group or warhead (covalently modifies an

active-site residue of the enzyme of interest)

Warhead linker tag

The reactive group of activity-based probe

* The reactive group is perhaps the most significant and difficult piece of the

probe to design.

It functions to covalently link the ABP to an amino acid residue

in the target enzyme’s active site when the target enzyme is active.

Nature chemical biology, Vol.1 No.3 August 2005

The reactive group of activity-based probe (2)

Nature chemical biology, Vol.1 No.3 August 2005

The mechanism of reactive group for enzyme targets

Chemical Reviews, Vol. 106, No.8, 2006

The tag region of activity-based probe

* The tag allows the identification or purification of modified enzymes.

Biotin, fluorescent small molecules, and radioactive isotopes are

most commonly incorporated into ABPs as tags

Current Opinion in Chemical Biology, Vol.11, 2007

The linker region of activity-based probe

* The linker region can be viewed as a bridge between the reactive

group and the labeling tag.

The linker serves to prevent steric hindrance by the tag that could

inhibit the reactivity of the probe, a linker can take the form of an

extended alkyl or polyethylene glycol (PEG) spacer.

The linker can serve as a specificity factor enabling targeting of the probe to

a specific enzyme or class of enzymes. For example , to target proteases,

this specificity region can be engineered to contain peptide sequences.

Application of Activity-Based Probes with affinity tag

* Identification of Biomarkers for Human Disease

Am. J. Pharmacogenomics , Vol.4, No.6 2004

Application of Activity-Based Probes with fluorescent tags

* In Vivo Imaging of Enzyme Activities

Am. J. Pharmacogenomics , Vol.4, No.6 2004

Competition of Activity-Based Probes with inhibitors

* Small Molecule Screening and Target Discovery

Am. J. Pharmacogenomics , Vol.4, No.6 2004

Case-discussion

* p90 ribosomal protein S6 kinases

RSK and MSK in MAP kinase signalling

• RSK (Ribosomal protein S6 Kinase) and MSK (Mitogen- and Stress- activated protein Kinase) constitute a family of protein kinases that mediate signal transduction downstream of MAP kinase cascades.

• RSK is activated by MAP kinases of the extracellular signalregulated kinase (ERK) family in response to growth factors, many polypeptide hormones, neurotransmitters, chemokines and other stimuli.

The domain structure and activation of RSK

Domain structure Activation and inactivation

• The N-terminal kinase domain (NTK) belongs to the AGC kinase family and is responsible for phospho

rylation of substrates.

• The C-terminal kinase domain (CTK) belongs to the CamK family and its only known function is activat

ion of NTK.

*AGC:containing PKA, PKG, PKC kinases family *CamK: calmodulin-dependent protein kinase family

J. Cell Sci. 119, 3021–3023 (2006)

Structural bioinformatics-based design of selective, irreversible RSK inhibitors

• All kinase inhibitors target the adenosine triphosphate (ATP) binding site

• The ATP binding sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. Structural bioinformatics approach to identify two selectivity filters: a threonine and a cysteine, at defined positions in the active site of p90 ribosoma lprotein S6 kinase (RSK)

Science 308, 1318–1321 (2005)

Unique design targeting non-conserved regions

Previous targeting strategy on ATP-binding site

Structural bioinformatics-based design of selective, irreversible RSK inhibitors

Science 308, 1318–1321 (2005)

• Selectivity filter 1: compact gatekeeper---Threonine

allows bulky aromatic substituents, such as those found in the Src family kinase inhibitors, PP1 and PP2, to enter a deep hydrophobic pocket

as ~20% of human kinases have a threonine at this position

• Selectivity filter 2: chemical reactive amino acid--- Cysteine

Out of 491 related kinase domains in the human genome, there are 11 kinase with a cysteine at the C-terminal end of the glycine-rich loop

A cysteine near this solvent exposed loop is likely to have a lower pKa and therefore to be more reactive than a cysteine buried in the hydrophobic pocket

RSK inhibitor

X

Structural bioinformatics-based design of selective, irreversible RSK inhibitors

Reactive group:

Fluoromethylketone (fmk)

Chloromethylketone (cmk)

p-tolyl substituent

Hydrophobic packet

Reactive cysteine

Science 308, 1318–1321 (2005)

In vitro assay (for RSK2)

* IC50 in uM

Cell-based assay (for HEK-293 cell)

* EC50 of ~150 nM

RSK RSK

PMA: Phorbol Myristate Acetate

The design of an fmk derivative (1)

Oncogene 25, 5764–5776 (2006)

* EC50 of >10 uM

Fluorescent tag

The design of an fmk derivative (2)

* Click chemistry method

Chemistry & Biology. 11, 535-546 (2004)

The design of an fmk derivative (2)

A clickable inhibitor for RSK

* EC50 of ~30 nM

• TAMRA is a fluorescent azide

Discussion

• fmk-pa, a propargylamine variant that has improved cellular potency and a ‘clickable’ tag for assessing the extent and selectivity of covalent RSK modification.

• Clickable inhibitors such as fmk-pa should facilitate determination of the specific roles played by the RSK CTD in cellular and animal models relevant to heart failure and other human diseases.

• Saturating concentrations of fmk-pa inhibited Ser386 phosphorylation and downstream signaling in response to phorbol ester stimulation, but had no effect on RSK activation by lipopolysaccharide.

Case-discussion (2)

* Metalloprotease

Metalloproteases are a large, diverse class of

enzymes involved in many physiological and

disease processes.

Metalloprotease (requiring activator)

• Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization .

inactive precursor enzyme (zymogens)

endogenous binding proteins (TIMPs)

• For cysteine protease : Acyloxy methyl ketone (AOMK) group

AOMK

Activity-based Probes Design of Metalloprotease

Metalloprotease

L L• For metalloprotease : do not use a catalytic amino acid side chain as the primary nucleophile catalytic zinc ion

PNAS 101, 10000-10005 (2004)

Hydroxamate (Hx) group

Hx: zinc-chelating group

(non-covalent bond)

Activity-based Probes Design of Metalloprotease

• First generation metalloproteases ABPs:

Hx group

benzophenone (BP): photo-cross-linker (for covalent bond formation)

O

hvO

E-CH2-

HO CH2-E

H2O

Rhodamine

Activity-based Probes Design of Metalloprotease

• New generation metalloproteases ABPs:

The large reporter tag which might be expected to obstruct

interactions with certain metalloproteases

Click chemistry method

Chemistry & Biology. 11, 535-546 (2004)

Activity-based Probes Synthesis of Metalloprotease

* General structure of the alkyne-tagged hydroxamate-benzophenone (HxBPyne) probe

Proteomic profiling of the HxBPyne probe library

• Mouse liver proteome

Proteomic profiling of the HxBPyne probe library

* Recombination expression sample

(breast cancer)

Proteomic profiling of the HxBPyne probe library

Proteomic profiling of the HxBPyne probe library

• 4 µg/ml of MMP in a background of 1 mg total protein / ml

• 1 µM probe• 1 uM LeuR2 HxBPyne

* Detection limit

Profiling Metalloproteases Activities by ABPP-MudPIT

• ABPP-MudPIT : Activity-Based Protein Profiling with Multidimensional

Protein Identification Technology

For enhancement of resolution and sensitivity

MudPIT

MudPIT

Nat. Bioltechnol. 19, 242-247 (2001)

Sensitivity of Detection of MMPs by ABPP-MudPIT

• 100 nM of the LeuR2 HxBPyne probe and analyzed by ABPP-MudPIT

• C: LeuR2 HxBPane (control)• detection limit 0.001~0.01% ( 5~50 fold)

Profiling Metalloprotease Activities in Cancer proteome

• To identify endogenous metalloprotease activities and quantify their relative levels in disease states the optimal probe set (cocktail): 100 nM of each HxBPyne probe; total 400 nM total probe

*C: 100 HxBPane competitor probes

Invasive Non-Invasive melanoma

Profiling Metalloprotease Activities in Cancer proteome

Discussion (2)

• ABPP may facilitate the simultaneous discovery of enzyme

activities associated with human disease and chemical tools

for testing their function in pathological processes

Quencher

Case-discussion (3)

* Cysteine protease: cathepsins

Papain-family protease (cathepsin B and L)

•Elevated cathepsin enzyme activity in serum or the extracellular matrix often

signifies a number of gross pathological conditions.

•Cathepsins are usually characterised as members of the lysosomal cysteine

protease (active site) family.

•Cathepsin-mediated diseases include: Alzheimer's, numerous types of cancer,

autoimmune related diseases like arthritis and the accelerated breakdown of

bone structure seen with osteoporosis

Chemical Reviews, 2002, Vol. 102, No. 12

Cysteine cathepsins in human cancer

Biol. Chem., Vol. 385, pp. 1017–1027, November 2004

Synthesis of the qABP GB117 and the control ABP GB111

FK

BODIPY ( tag)

Phenylalanine-lysine dipeptide

(linker)

ABP

AOMK

AOMK: Acyloxy methyl ketone

(warhead)

2,6-dimethyl benzoic acid AOMK

N-protected glycine AOMK

Synthesis of the qABP GB117 and the control ABP GB111

G

QSY7 (quencher)

qABP

BODIPY ( tag)

QSY7 (quencher)

Determination of quenching efficiency of qABP GB117 relative to the unquenched control GB111

LysoTracker (lysosomal marker): Weakly basic amines selectively

accumulate in cellular compartments with low internal pH and can

be used to investigate the biosynthesis and pathogenesis of lysosomes

Structure of the new qABPs (NIRF-ABPs)

The most stable probe

Labeling of recombinant cathepsins and intact cells with the control ABP and qABP

* NIH-3T3 cells

* Inhibitor: GB111-NH2

O

O

OHN

NH

O

O

O

NH2

Non-

specific

Labeling of recombinant cathepsins and intact cells with the control ABP and qABP (2)

* Inhibitor: GB111-NH2

O

O

OHN

NH

O

O

O

NH2

Optical imaging of tumors in live mice using non-quenched NIRF-ABPS

GB 123

GB 123

GB 138

GB 125

CCD camera-based imaging system

(Xenogen IVIS200 imaging system)

Biochemical characterization of in vivo-labeled proteases

The signals observed in the live animals were due to specific modification of active cysteine cathepsins.

Direct comparison of the non-quenched and quenched NIRF-ABPs

The quenched probe achieved its maximum much more rapidly than the non-quenched probe

Imaging of in vivo efficacy of small-molecule inhibitors

N

N

O

NH

O

HN S

OO

Inhibitor: K11777

Discussion (3)•To developed a new class of qNIRF-ABPs that become fluorescent upon

activity-dependent covalent modification of a protease target.

•These probes allow direct in vivo analysis of drug efficacy and pharmacodynamic

properties.

•The NIRF-ABPs that allow the activity of the cysteine cathepsins B and L to

be visualized in living subjects.

•A current limitation of this technology is that it is only applicable to superficial

tissues, and the high levels of signal in large organs with high cathepsin

activity such as liver, kidney and spleen make imaging of specific locations

within the central body cavity difficult.

Conclusion

We would like to emphasize that the field of activity-based probe has a great

potential of significantly advancing our understanding of biology by elucidation

of protein function and also to speed up drug development in the future.

Nature Chemical Biology 2, 689-700 (2006)