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BioAssay: AID 743257

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

Name: QFRET-based biochemical high throughput confirmation assay to identify exosite inhibitors of ADAM17. ..more
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AID: 743257
Data Source: The Scripps Research Institute Molecular Screening Center (ADAM17_INH_QFRET_1536_3X%INH CRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2014-01-17

Data Table ( Complete ):           Active    All
BioActive Compounds: 1855
Depositor Specified Assays
720648QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM17.screeningPrimary Assay (ADAM17 INH in singlicate)
743013Summary of the probe development effort to identify exosite inhibitors of ADAM17.summarySummary
743259Counterscreen for exosite inhibitors of ADAM17: Fluorescence resonance energy transfer (FRET)-based biochemical high throughput dose response assay to identify inhibitors of ADAM10confirmatory
743260QFRET-based biochemical high throughput dose response assay to identify exosite inhibitors of ADAM17confirmatory
743337On Hold
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_3X%INH CRUN

Name: QFRET-based biochemical high throughput confirmation 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[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].


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CRUN, confirm, confirmation, triplicate, 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.
Assay Overview:

The purpose of this assay is to confirm ADAM17 inhibitory activity of compounds identified as active in a set of previous experiments entitled, "QFRET-based biochemical primary high throughput screening assay to identify exosite inhibitors of ADAM17." (AID 720648). 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.

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


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.

Interval Cutoff:

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 720648) 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. The MLSMR was not able to provide all compounds selected for testing in this assay.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 6.95 uM (6.95μM**)Normalized percent inhibition of the primary screen at a compound concentration of 6.95 micromolar.Float%
2Standard DeviationStandard deviation derived from the normalized percent inhibition of the triplicate data for each compoundFloat
3Inhibition at 6.95 uM [1] (6.95μM**)Percent inhibition of the confirmatory screen at a compound concentration of 6.95 micromolar.Float%
4Inhibition at 6.95 uM [2] (6.95μM**)Percent inhibition of the confirmatory screen at a compound concentration of 6.95 micromolar.Float%
5Inhibition at 6.95 uM [3] (6.95μM**)Percent inhibition of the confirmatory screen at a compound concentration of 6.95 micromolar.Float%

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

Data Table (Concise)