QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM17.
Name: QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM17. ..more
BioActive Compounds: 3080
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: ADAM17_INH_QFRET_1536_1X%INH PRUN
Name: QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM17.
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.
PRUN, primary, singlicate, 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 assay is to identify compounds that inhibit ADAM17. 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 singlicate at a final nominal concentration of 6.95 micromolar.
Prior to the start of the assay, 2.5 microliters 2X ADAM17 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.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-8, for inactive 8-0.
List of Reagents:
ADAM17 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. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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, 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)