External ID: AlphaScreenDoseResponseEnamine68
Protocol:
SSBct-PriA AlphaScreen Assay
Screening took place at the University of Wisconsin Small Molecule Screening Facility (SMSF). Controls and small molecules (stored as 10 mM DMSO stocks) were dispensed into 1536-well white plates (Nunc 253607, ThermoFisher) using an Echo 550 (Labcyte, Sunnyvale, CA) acoustic liquid handler. A Mantis liquid handler equipped with a high-volume silicone chip (Formulatrix, Bedford, MA) was used to add 3.0 muL of a master mix containing 10 mM HEPES-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 10 mM DTT, 1 mg/mL bovine serum albumin (BSA), 0.01% Triton X-100, 0.1 micro M PriA, 0.1 micro M Bio-SSBct, and 5 microgram/mL AlphaScreen (AS) of both donor and acceptor beads (PerkinElmer, Waltham, MA) to each well of the plate. Master mix was prepared under diminished lighting immediately prior to dispensing. Plates were centrifuged briefly, rocked at room temperature for an hour, and then read using a PheraStar (BMG Labtech, Offenburg, Germany) plate reader using the following settings: 0.1 s settling time, 0.3 s excitation, and 0.6 s integration time with a 0.04 s delay between excitation and integration. Final concentrations of the positive (SSBct) and negative (DeltaFSSBct) controls were 25 micro M. Each screening plate contained 32 positive and negative control wells, 43 DMSO-only control wells, and a control SSBct concentration-response curve conducted in triplicate with SSBct concentrations of 0.5, 1, 2, 4, 8, 16, and 32 muM. For compound concentration-response curves, the requisite amount of each compound (at 10 mM in DMSO) was added to the wells and then backfilled with 100% DMSO so that each well contained a final DMSO concentration of 0.33% (v/v). AS master mix was then added to each well as before.
All 68 compounds were initially screened in four replicates at eight concentrations ranging from 515.6 nm to 66 microm. We defined compounds whose median % inhibition at 33 microm was at least 50%, the same threshold used for the AMS screen (AID 1272365), as initial hits. We repeated the dose-response curve screens for two additional rounds of ten compounds each, expanding the range of concentrations tested to improve the quality of the curve fits. We defined confirmed hits (actives) as those with a dose-response curve in curve class (Inglese et al., 2006) 1.2 or in curve class 2.2 with an IC50 95% upper confidence limit within the tested range of concentrations. We used the Collaborative Drug Discovery Vault software (Ekins and Bunin, 2013) to fit dose-response curves, calculate IC50 values and the 95% upper and lower confidence limits, and define curve classes. For curve classifications, see https://support.collaborativedrug.com/hc/en-us/articles/214359343-Activity-and-Curve-Validation#curve_classification. Well-defined CIs were only achieved on compounds having IC50 <= 40 micromolar in the 4PL curve fits.
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