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BioAssay: AID 743260

QFRET-based biochemical high throughput dose response assay to identify exosite inhibitors of ADAM17

Name: QFRET-based biochemical high throughput dose response assay to identify exosite inhibitors of ADAM17. ..more
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 Tested Compounds
 Tested Compounds
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Active(5)
 
 
Inactive(243)
 
 
 Tested Substances
 Tested Substances
All(248)
 
 
Active(5)
 
 
Inactive(243)
 
 
AID: 743260
Data Source: The Scripps Research Institute Molecular Screening Center (ADAM17_INH_QFRET_1536_3XIC50 INH DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2014-01-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5
Related Experiments
AIDNameTypeComment
720648QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM17.Screeningdepositor-specified cross reference: Primary Assay (ADAM17 INH in singlicate)
743013Summary of the probe development effort to identify exosite inhibitors of ADAM17.Summarydepositor-specified cross reference: Summary
743256Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput screening assay to identify inhibitors of ADAM10Screeningdepositor-specified cross reference: Counterscreen Assay (ADAM10 INH in triplicate)
743257QFRET-based biochemical high throughput confirmation assay to identify exosite inhibitors of ADAM17Screeningdepositor-specified cross reference: Confirmation Assay (ADAM17 INH in triplicate)
743259Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput dose response assay to identify inhibitors of ADAM10Confirmatorysame project related to Summary assay
743337On Hold
Description:
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_3XIC50 INH DRUN

Name: QFRET-based biochemical high throughput dose response assay to identify exosite inhibitors of ADAM17.

Description:

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[12]. 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].

References:

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.

Keywords:

DRUN, 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.
Protocol
Assay Overview:
The purpose of this biochemical assay is to determine ADAM17 inhibitory dose response curves of compounds identified as active in a previous set experiments entitled "QFRET-based biochemical high throughput confirmation assay to identify exosite inhibitors of ADAM17". Specifically, this assay is used 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 triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 69.5 uM.
Protocol Summary:
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
Where:
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:
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)
Comment
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 [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Biochemical
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Qualifierinhibition Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the inhibition in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
8RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
9Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
10Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
11Response RangeThe range of Y.Float
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.0035 uM [1] (0.0035μM**)Value of % inhibition at 0.0035 uM compound concentration; replicate [1]Float%
14Inhibition at 0.0035 uM [2] (0.0035μM**)Value of % inhibition at 0.0035 uM compound concentration; replicate [2]Float%
15Inhibition at 0.0035 uM [3] (0.0035μM**)Value of % inhibition at 0.0035 uM compound concentration; replicate [3]Float%
16Inhibition at 0.011 uM [1] (0.011μM**)Value of % inhibition at 0.011 uM compound concentration; replicate [1]Float%
17Inhibition at 0.011 uM [2] (0.011μM**)Value of % inhibition at 0.011 uM compound concentration; replicate [2]Float%
18Inhibition at 0.011 uM [3] (0.011μM**)Value of % inhibition at 0.011 uM compound concentration; replicate [3]Float%
19Inhibition at 0.032 uM [1] (0.032μM**)Value of % inhibition at 0.032 uM compound concentration; replicate [1]Float%
20Inhibition at 0.032 uM [2] (0.032μM**)Value of % inhibition at 0.032 uM compound concentration; replicate [2]Float%
21Inhibition at 0.032 uM [3] (0.032μM**)Value of % inhibition at 0.032 uM compound concentration; replicate [3]Float%
22Inhibition at 0.095 uM [1] (0.095μM**)Value of % inhibition at 0.095 uM compound concentration; replicate [1]Float%
23Inhibition at 0.095 uM [2] (0.095μM**)Value of % inhibition at 0.095 uM compound concentration; replicate [2]Float%
24Inhibition at 0.095 uM [3] (0.095μM**)Value of % inhibition at 0.095 uM compound concentration; replicate [3]Float%
25Inhibition at 0.29 uM [1] (0.29μM**)Value of % inhibition at 0.29 uM compound concentration; replicate [1]Float%
26Inhibition at 0.29 uM [2] (0.29μM**)Value of % inhibition at 0.29 uM compound concentration; replicate [2]Float%
27Inhibition at 0.29 uM [3] (0.29μM**)Value of % inhibition at 0.29 uM compound concentration; replicate [3]Float%
28Inhibition at 0.86 uM [1] (0.86μM**)Value of % inhibition at 0.86 uM compound concentration; replicate [1]Float%
29Inhibition at 0.86 uM [2] (0.86μM**)Value of % inhibition at 0.86 uM compound concentration; replicate [2]Float%
30Inhibition at 0.86 uM [3] (0.86μM**)Value of % inhibition at 0.86 uM compound concentration; replicate [3]Float%
31Inhibition at 2.6 uM [1] (2.6μM**)Value of % inhibition at 2.6 uM compound concentration; replicate [1]Float%
32Inhibition at 2.6 uM [2] (2.6μM**)Value of % inhibition at 2.6 uM compound concentration; replicate [2]Float%
33Inhibition at 2.6 uM [3] (2.6μM**)Value of % inhibition at 2.6 uM compound concentration; replicate [3]Float%
34Inhibition at 7.7 uM [1] (7.7μM**)Value of % inhibition at 7.7 uM compound concentration; replicate [1]Float%
35Inhibition at 7.7 uM [2] (7.7μM**)Value of % inhibition at 7.7 uM compound concentration; replicate [2]Float%
36Inhibition at 7.7 uM [3] (7.7μM**)Value of % inhibition at 7.7 uM compound concentration; replicate [3]Float%
37Inhibition at 23.2 uM [1] (23.2μM**)Value of % inhibition at 23.2 uM compound concentration; replicate [1]Float%
38Inhibition at 23.2 uM [2] (23.2μM**)Value of % inhibition at 23.2 uM compound concentration; replicate [2]Float%
39Inhibition at 23.2 uM [3] (23.2μM**)Value of % inhibition at 23.2 uM compound concentration; replicate [3]Float%
40Inhibition at 69.5 uM [1] (69.5μM**)Value of % inhibition at 69.5 uM compound concentration; replicate [1]Float%
41Inhibition at 69.5 uM [2] (69.5μM**)Value of % inhibition at 69.5 uM compound concentration; replicate [1]Float%
42Inhibition at 69.5 uM [3] (69.5μM**)Value of % inhibition at 69.5 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA033985-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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