Low volume 384 well plates

Armadillo PCR Plates, 384-well, were purchased from Thermo Fisher Scientific. Low-volume, 384-well plates were purchased from Corning. Horseradish Peroxidase, and the luminescent HRP substrate, SuperSignal™ West Femto Maximum Sensitivity Substrate, were also purchased from Thermo Fisher Scientific. PhosSTOP Inhibitor Tablets, Roche cOmplete™ mini EDTA-free Protease Inhibitor Tablets, tris(2-carboxyethyl)phosphine (TCEP), hexadecyl phosphonic acid (HDPA), ethylenediaminetetraacetic acid (EDTA), and 2,4,6-tri-hydroxyacetophenone (THAP) were purchased from Sigma-Aldrich. The phosphopeptide (Ac-IpYERC-NH₂) was synthesized using Fmoc solid phase as previously described.²⁶

AlphaScreen (Perkin Elmer, Waltham, MA) was utilized largely as previously described.¹⁸ Screening was performed in 1536 well plates (Nunc, Rochester, NY) with final concentrations of each component being 10 nM each protein (RGS and G protein) and 10 ng/µL each bead. The NCI NExT Diversity Library was screened at a final compound concentration of 35.7 µM. Due to financial constraints, only 60,502 compounds were screened from this library, representing roughly 75% coverage of the 83,536 compound collection. Dose response follow up was performed in 384 well low volume plates (Corning, Kennebunk, ME). Three point dose response assessment utilized compound final concentrations of 119 µM, 35.7 µM, and 11.9 µM. For nine point dose response assessment compound final concentrations ranged from 100 μM to 31.6 nM following a half log dilution pattern. Counter screening / assay interference control assay utilized the AlphaScreen TruHits assay (Perkin Elmer, Waltham, MA) as previously described.¹⁸ All plates were read on an Envision plate reader (Perkin Elmer, Waltham, MA) and all data were analyzed using GraphPad Prism 7 (GraphPad, La Jolla, CA).

LanthaScreen™ Eu time-resolved fluorescence resonance energy transfer (TR-FRET) kinase binding assays (Invitrogen) were performed in 384-well, low-volume plates (Corning) using recombinant WEE1 kinase, Kinase Tracer 178 and LanthaScreen™ Eu-anti-GST antibody (Invitrogen). Assays were performed at 25°C in a reaction mixture consisting of 5 μL serially diluted inhibitor solution, 5 μL Kinase Tracer 178 solution, and 5 μL kinase/antibody solution. All reagents were prepared as solutions in 1X kinase buffer A (Invitrogen) at 3X final desired concentration. Inhibitor solutions were prepared such that final DMSO concentrations did not exceed 0.5%, which was shown to have no effect on kinase activity. Inhibitors were assayed in the final concentration range of 0.04 nm to 10 μm. Kinase Tracer 178 was used at a final concentration of 150 nm and the antibody and kinase were used at final concentrations of 3 nm and 5 nm, respectively. All reagents were incubated together for 1 h at room temperature and read using a PerkinElmer Envision 2104 Multilabel Reader enabled for TR-FRET (Excitation = 340 nm; Tracer emission = 665 nm; Antibody emission = 615 nm; Delay = 100 μs; Integration = 200 μs). Emission ratios (665 nm/615 nm) were determined for each inhibitor concentration and the data analyzed using a non-linear regression analysis of the log dose-response curve to determine IC₅₀ values.