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
BioActive Compounds: 5
Depositor Specified Assays
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.
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].
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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.
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.
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.
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)
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.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)