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

Application of Activity-Based Probes with fluorescent tags * In Vivo Imaging of Enzyme Activities Am. J. Pharmacogenomics, Vol.4, No

Competition of Activity-Based Probes with inhibitors * Small Molecule Screening and Target Discovery Am. J. Pharmacogenomics, Vol.4, No

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 structureActivation and inactivation The N-terminal kinase domain (NTK) belongs to the AGC kinase family and is responsible for phosphorylation of substrates. The C-terminal kinase domain (CTK) belongs to the CamK family and its only known function is activation 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) * IC 50 in uM Cell-based assay (for HEK-293 cell) * EC 50 of ~150 nM RSK PMA: Phorbol Myristate Acetate

The design of an fmk derivative (1) Oncogene 25, 5764–5776 (2006) * EC 50 of >10 uM Fluorescent tag

The design of an fmk derivative (2) * Click chemistry method Chemistry & Biology. 11, (2004)

The design of an fmk derivative (2)

A clickable inhibitor for RSK * EC 50 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, (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) 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, (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

4 µg/ml of MMP in a background of 1 mg total protein / ml 1 µM probe 1 uM LeuR 2 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 Nat. Bioltechnol. 19, (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 InvasiveNon-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 F K 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-NH 2 Non- specific

Labeling of recombinant cathepsins and intact cells with the control ABP and qABP (2) * Inhibitor: GB111-NH 2

Optical imaging of tumors in live mice using non- quenched NIRF-ABPS 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 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, (2006)