Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput dose response assay to identify inhibitors of ADAM10
Name: Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput dose response assay to identify inhibitors of ADAM10. ..more
BioActive Compounds: 28
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: Torrey Pines Institute for Molecular Sciences (TPIMS)
Assay Provider: Dmitriy Minond, Torrey Pines Institute for Molecular Sciences (TPIMS)
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA033985-01
Grant Proposal PI: Dmitriy Minond, Torrey Pines Institute for Molecular Sciences (TPIMS)
External Assay ID: ADAM10_INH_QFRET_1536_3XIC50 INH DCSRUN for ADAM17 INH
Name: Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput dose response assay to identify inhibitors of ADAM10.
Approximately 20-30% of breast cancer patients have tumors that over-express human epidermal growth factor receptor (HER2), which confers an aggressive tumor phenotype and poor prognosis [1-3]. A Disintegrin and Metalloprotease (ADAM) proteases are responsible for amplification of HER2 signaling due to either cleavage of its extracellular domain or release of HER2 ligands, which leads to proliferation and inhibition of apoptosis [4, 5]. ADAM proteases implicated in amplification of HER2 signaling [6, 7] are ADAM10 [8, 9] and 17 [10, 11]; therefore, inhibition of these proteases represents a viable approach to the abrogation of HER2 signaling in breast cancer. The specific aims of this proposal, therefore, will focus on (1) screening of the MLPCN library for inhibitors that interact with exosites of ADAM10 and 17, and (2) medicinal chemistry optimization of initial leads in order to develop molecular probes of ADAM10 and 17. Our laboratory is uniquely positioned to achieve these goals due to expertise in development of exosite-binding inhibitors and probes, HTS, and biochemistry of proteases. We will also collaborate with experts in the fields of peptide synthesis, HTS, and medicinal chemistry. The successful completion of the Aims of this proposal will lead to a discovery of novel, potent, and selective small molecule probes for ADAM10 and 17. Using these selective molecular probes alone or in combination with other tools, such as siRNA, antibodies, and other small molecule inhibitors, the researchers will be able to study contributions not only of individual members of the ADAM protease family, but also the interplay of ADAM protease-controlled pathways with other pathways implicated in the progression of breast cancer [13-15].
1. CDC, United States Cancer Statistics: Incidence and Mortality Web-based Report: 1999-2007 Cancer Incidence and Mortality Data. 2010, Department of Health and Human Services: Atlanta.
2. Steger, G.G., J. Abrahamova, F. Bacanu, S. Brincat, A. Brize, A. Cesas, T. Cufer, M. Dank, R. Duchnowska, A. Eniu, J. Jassem, Z. Kahan, E. Matos, P. Padrik, S. Plate, H. Pokker, G. Purkalne, C. Timcheva, V. Tzekova, R. Vyzula, and C.C. Zielinski, Current standards in the treatment of metastatic breast cancer with focus on Lapatinib: a review by a Central European Consensus Panel. Wien Klin Wochenschr, 2010. 122(11-12): p. 368-79.
3. Barros, F.F., D.G. Powe, I.O. Ellis, and A.R. Green, Understanding the HER family in breast cancer: interaction with ligands, dimerization and treatments. Histopathology, 2010. 56(5): p. 560-72.
4. Klein, T. and R. Bischoff, Active metalloproteases of the A Disintegrin and Metalloprotease (ADAM) family: biological function and structure. J Proteome Res, 2011. 10(1): p. 17-33.
5. Mazzocca, A., G. Giannelli, and S. Antonaci, Involvement of ADAMs in tumorigenesis and progression of hepatocellular carcinoma: Is it merely fortuitous or a real pathogenic link? Biochim Biophys Acta, 2010. 1806(1): p. 74-81.
6. Gijsen, M., P. King, T. Perera, P. Parker, B. Larijani, A. Harris, and A. Kong, Upregulation of ADAM proteases and HER ligands through a feedback loop mediates acquired resistance to trastuzumab in HER2-amplified breast cancer. Breast Cancer Res, 2010. 12 Suppl 1: p. O2.
7. Blobel, C.P., ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol, 2005. 6(1): p. 32-43.
8. Gibb, D.R., S.J. Saleem, N.S. Chaimowitz, J. Mathews, and D.H. Conrad, The emergence of ADAM10 as a regulator of lymphocyte development and autoimmunity. Mol Immunol, 2011. 48(11): p. 1319-27.
9. Endres, K. and F. Fahrenholz, Upregulation of the alpha-secretase ADAM10--risk or reason for hope? FEBS J, 2010. 277(7): p. 1585-96.
10. Scheller, J., A. Chalaris, C. Garbers, and S. Rose-John, ADAM17: a molecular switch to control inflammation and tissue regeneration. Trends Immunol, 2011. 32(8): p. 380-7.
11. Gooz, M., ADAM-17: the enzyme that does it all. Crit Rev Biochem Mol Biol, 2010. 45(2): p. 146-69.
12. Sinnathamby, G., J. Zerfass, J. Hafner, P. Block, Z. Nickens, A. Hobeika, A.A. Secord, H.K. Lyerly, M.A. Morse, and R. Philip, ADAM metallopeptidase domain 17 (ADAM17) is naturally processed through major histocompatibility complex (MHC) class I molecules and is a potential immunotherapeutic target in breast, ovarian and prostate cancers. Clin Exp Immunol, 2011. 163(3): p. 324-32.
13. Duffy, M.J., M. Mullooly, N. O'Donovan, S. Sukor, J. Crown, A. Pierce, and P.M. McGowan, The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer? Clin Proteomics, 2011. 8(1): p. 9.
14. Gijsen, M., P. King, T. Perera, P.J. Parker, A.L. Harris, B. Larijani, and A. Kong, HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer. PLoS Biol, 2010. 8(12): p. e1000563.
15. Liu, X., J.S. Fridman, Q. Wang, E. Caulder, G. Yang, M. Covington, C. Liu, C. Marando, J. Zhuo, Y. Li, W. Yao, K. Vaddi, R.C. Newton, P.A. Scherle, and S.M. Friedman, Selective inhibition of ADAM metalloproteases blocks HER-2 extracellular domain (ECD) cleavage and potentiates the anti-tumor effects of trastuzumab. Cancer Biol Ther, 2006. 5(6): p. 648-56.
16. Kenny, P.A. and M.J. Bissell, Targeting TACE-dependent EGFR ligand shedding in breast cancer. J Clin Invest, 2007. 117(2): p. 337-45.
17. Moss, M.L., L. Sklair-Tavron, and R. Nudelman, Drug insight: tumor necrosis factor-converting enzyme as a pharmaceutical target for rheumatoid arthritis. Nat Clin Pract Rheumatol, 2008. 4(6): p. 300-9.
18. Georgiadis, D. and A. Yiotakis, Specific targeting of metzincin family members with small-molecule inhibitors: progress toward a multifarious challenge. Bioorg Med Chem, 2008. 16(19): p. 8781-94.
19. Edwards, D.R., M.M. Handsley, and C.J. Pennington, The ADAM metalloproteinases. Mol Aspects Med, 2008. 29(5): p. 258-89.
20. Johnson, W.H., N.A. Roberts, and N. Borkakoti, Collagenase inhibitors: their design and potential therapeutic use. J Enzyme Inhib, 1987. 2(1): p. 1-22.
21. Dennis, M.S., C. Eigenbrot, N.J. Skelton, M.H. Ultsch, L. Santell, M.A. Dwyer, M.P. O'Connell, and R.A. Lazarus, Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature, 2000. 404(6777): p. 465-70.
22. Roberge, M., M. Peek, D. Kirchhofer, M.S. Dennis, and R.A. Lazarus, Fusion of two distinct peptide exosite inhibitors of Factor VIIa. Biochem J, 2002. 363(Pt 2): p. 387-93.
23. Roberge, M., L. Santell, M.S. Dennis, C. Eigenbrot, M.A. Dwyer, and R.A. Lazarus, A novel exosite on coagulation factor VIIa and its molecular interactions with a new class of peptide inhibitors. Biochemistry, 2001. 40(32): p. 9522-31.
24. Izaguirre, G., A.R. Rezaie, and S.T. Olson, Engineering functional antithrombin exosites in alpha1-proteinase inhibitor that specifically promote the inhibition of factor Xa and factor IXa. J Biol Chem, 2009. 284(3): p. 1550-8.
25. Scheer, J.M., M.J. Romanowski, and J.A. Wells, A common allosteric site and mechanism in caspases. Proc Natl Acad Sci U S A, 2006. 103(20): p. 7595-600.
26. Johnson, A.R., A.G. Pavlovsky, D.F. Ortwine, F. Prior, C.F. Man, D.A. Bornemeier, C.A. Banotai, W.T. Mueller, P. McConnell, C. Yan, V. Baragi, C. Lesch, W.H. Roark, M. Wilson, K. Datta, R. Guzman, H.K. Han, and R.D. Dyer, Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects. J Biol Chem, 2007. 282(38): p. 27781-91.
27. Engel, C.K., B. Pirard, S. Schimanski, R. Kirsch, J. Habermann, O. Klingler, V. Schlotte, K.U. Weithmann, and K.U. Wendt, Structural basis for the highly selective inhibition of MMP-13. Chem Biol, 2005. 12(2): p. 181-9.
28. Baragi, V.M., G. Becher, A.M. Bendele, R. Biesinger, H. Bluhm, J. Boer, H. Deng, R. Dodd, M. Essers, T. Feuerstein, B.M. Gallagher, Jr., C. Gege, M. Hochgurtel, M. Hofmann, A. Jaworski, L. Jin, A. Kiely, B. Korniski, H. Kroth, D. Nix, B. Nolte, D. Piecha, T.S. Powers, F. Richter, M. Schneider, C. Steeneck, I. Sucholeiki, A. Taveras, A. Timmermann, J. Van Veldhuizen, J. Weik, X. Wu, and B. Xia, A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models. Arthritis Rheum, 2009. 60(7): p. 2008-18.
29. Lauer-Fields, J.L., D. Minond, P.S. Chase, P.E. Baillargeon, S.A. Saldanha, R. Stawikowska, P. Hodder, and G.B. Fields, High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate. Bioorg Med Chem, 2009. 17(3): p. 990-1005.
DCSRUN, counterscreen, dose response, triplicate, end point, end-point, titration, dilution, exosite, ADAM, ADAM17, TACE, protease, enzyme, fluorescence, FRET, binding, cancer, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this bichemical assay is to determine ADAM10 inhibitory dose response curves for compounds that confirmed activity in a set of previous experiments entitled: of compounds identified as active in a set of previous experiments entitled "Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput screening assay to identify inhibitors of ADAM10". This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 69.5 uM.
Prior to the start of the assay, 2.5 microliters 2X ADAM10 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).
The % inhibition for each well was then calculated as follows:
%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells treated with 1 micromolar Marimastat
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 69.5 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 69.5 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
List of Reagents:
ADAM10 enzyme (R&D Systems, part # 930-ADB)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (Corning, part # 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
Assay: Dictionary: Version: 0.1
Assay Format: Biochemical
Assay Type: Binding
* Activity Concentration. ** Test Concentration.
Data Table (Concise)