Presentation Transcript

1. Activity-Based Protein Profiling

2. 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
3. Assignment of protein function in the postgenomic era * In the postgenomic era
researchers are now confronted with the task of assigning functions to tens of
thousands of proteins. 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. Global analysis of changes in gene
transcription and translation by abundance-based genomic and proteomic
approaches provides only indirect information about protein function. 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.
4. 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 Examples (3
becomes more popular now) 1. Small-molecule substrate reporters of enzymatic
activity  2. Protein-based reporters of enzymatic activity  3. Activity-based probe
5. 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 Disadvantages: 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

6. Protein-based reporters of enzymatic activity Fluorescent reporters

Bioluminescent reporters Disadvantages: 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
7. What Is an Activity-Based probe (ABP) ? * The activity-based probes (ABPs): they
generally contain three main functional groups: 1. The chemical reactive group or
warhead (covalently modifies an active-site residue of the enzyme of interest) 2. A
linker region, which can be specific for different enzymes 3. A tag, which is used to
visualize the modified enzyme Warhead linker tag
8. 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
9. The reactive group of activity-based probe (2) Nature chemical biology, Vol.1 No.3
August 2005
10. The mechanism of reactive group for enzyme targets Chemical Reviews, Vol. 106,
No.8, 2006
11. 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
12. 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.
13. Application of Activity-Based Probes with affinity tag * Identification of
Biomarkers for Human Disease Am. J. Pharmacogenomics , Vol.4, No.6 2004
14. Application of Activity-Based Probes with fluorescent tags * In Vivo Imaging of
Enzyme Activities Am. J. Pharmacogenomics , Vol.4, No.6 2004
15. Competition of Activity-Based Probes with inhibitors * Small Molecule Screening
and Target Discovery Am. J. Pharmacogenomics , Vol.4, No.6 2004
16. Case-discussion * p90 ribosomal protein S6 kinases
17. 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.
18. 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 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)
19. Structural bioinformatics-based design of selective, irreversible RSK inhibitors
Previous targeting strategy on ATP-binding site • 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) Unique design targeting non-
conserved regions Science308, 1318–1321 (2005)
20. RSK inhibitor X Structural bioinformatics-based design of selective, irreversible RSK
inhibitors • 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 Science308, 1318–
1321 (2005)
21. p-tolyl substituent Reactive group: Fluoromethylketone (fmk) Chloromethylketone
(cmk) RSK RSK Cell-based assay (for HEK-293 cell) In vitro assay (for RSK2) PMA:
Phorbol Myristate Acetate * IC50 in uM * EC50 of ~150 nM Structural
bioinformatics-based design of selective, irreversible RSK inhibitors Hydrophobic
packet Reactive cysteine Science308, 1318–1321 (2005)
22. Fluorescent tag The design of an fmk derivative (1) * EC50 of >10 uM Oncogene25,
5764–5776 (2006)
23. * Click chemistry method Chemistry & Biology. 11, 535-546 (2004) The design of
an fmk derivative (2)
24. The design of an fmk derivative (2)
25. A clickable inhibitor for RSK TAMRA is a fluorescent azide * EC50 of ~30 nM
26. 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. • 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. • 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.
27. Case-discussion (2) * Metalloprotease  Metalloproteases are a large, diverse class
of enzymes involved in many physiological and disease processes.
28. 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)
29. AOMK • For cysteine protease : Acyloxy methyl ketone (AOMK) group
30. L L Metalloprotease Hydroxamate (Hx) group Hx: zinc-chelating group (non-
covalent bond) Activity-based Probes Design of Metalloprotease • For
metalloprotease : • do not use a catalytic amino acid side chain as the primary
nucleophile • catalytic zinc ion PNAS 101, 10000-10005 (2004)
31. Rhodamine Hx group benzophenone (BP): photo-cross-linker (for covalent bond
formation) Activity-based Probes Design of Metalloprotease • First generation
metalloproteases ABPs:
32.  Click chemistry method Chemistry & Biology. 11, 535-546 (2004) Activity-based
Probes Design of Metalloprotease • New generation metalloproteases ABPs:  The
large reporter tag which might be expected to obstruct interactions with certain
metalloproteases
33. * General structure of the alkyne-tagged hydroxamate-benzophenone (HxBPyne)
probe Activity-based Probes Synthesis of Metalloprotease
34. Mouse liver proteome Proteomic profiling of the HxBPyne probe library
35. Proteomic profiling of the HxBPyne probe library * Recombination expression
sample (breast cancer)
36. Proteomic profiling of the HxBPyne probe library
37. 4 µg/ml of MMP in a background of 1 mg total protein / ml 1 µM probe Proteomic
profiling of the HxBPyne probe library * Detection limit 1 uM LeuR2 HxBPyne
38. MudPIT 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, 242-247 (2001)
39. 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)
40. 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 Invasive Non-Invasive melanoma *C: 100 HxBPane competitor probes
41. Profiling Metalloprotease Activities in Cancer proteome
42. 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
43. Quencher Case-discussion (3) * Cysteine protease: cathepsins
44. Cathepsins are usually characterised as members of the lysosomal cysteine
protease (active site) family. • Elevated cathepsin enzyme activity in serum or the
extracellular matrix often signifies a number of gross pathological conditions. •
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 Papain-family protease (cathepsin B and L)
Chemical Reviews, 2002, Vol. 102, No. 12
45. Cysteine cathepsins in human cancer Biol. Chem., Vol. 385, pp. 1017–1027,
November 2004
46. 2,6-dimethyl benzoic acid AOMK N-protected glycine AOMK AOMK: Acyloxy
methyl ketone (warhead) F K BODIPY ( tag) AOMK Synthesis of the qABP GB117
and the control ABP GB111 Phenylalanine-lysine dipeptide (linker) ABP
47. BODIPY ( tag) G QSY7 (quencher) QSY7 (quencher) Synthesis of the qABP GB117
and the control ABP GB111 qABP
48. 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
49. Structure of the new qABPs (NIRF-ABPs) The most stable probe
50. * Inhibitor: GB111-NH2 Labeling of recombinant cathepsins and intact cells with
the control ABP and qABP Non- specific * NIH-3T3 cells
51. * Inhibitor: GB111-NH2 Labeling of recombinant cathepsins and intact cells with
the control ABP and qABP (2)
52. GB 123 GB 123 GB 138 GB 125 Optical imaging of tumors in live mice using non-
quenched NIRF-ABPS CCD camera-based imaging system (Xenogen IVIS200 imaging
system)
53. Biochemical characterization of in vivo-labeled proteases The signals observed in
the live animals were due to specific modification of active cysteine cathepsins.
54. Direct comparison of the non-quenched and quenched NIRF-ABPs The quenched
probe achieved its maximum much more rapidly than the non-quenched probe
55. Imaging of in vivo efficacy of small-molecule inhibitors Inhibitor: K11777
56. Discussion (3) • To developed a new class of qNIRF-ABPs that become fluorescent
upon activity-dependent covalent modification of a protease target. • The NIRF-
ABPs that allow the activity of the cysteine cathepsins B and L to be visualized in
living subjects. • These probes allow direct in vivo analysis of drug efficacy and
pharmacodynamic properties. • 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.
57. 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)