Multidrop 384

The high-throughput screening of 210,000 compounds was conducted in 384-well plates using Kinase-Glo ^® (Promega Inc.). Seven hundred and forty-one plates were screened, 36 plates per day, in the UT Southwestern High-Throughput Screening Core (HTS). The substrate solution was comprised of 55.3 mM HEPES (pH 7.4), 55.3 mM MgCl₂, 4.7 µM pWNK1, and 19.3 µM GST-OSR1. Fifteen microliters of it was added to each well by multiplexed dispensing (Multidrop384, Thermo Scientific). Then, 0.2 µL of pure DMSO (negative control, columns 2 and 23) or 0.5 mM compound (columns 3 to 22) or 0.4 mM control quinazoline inhibitor (Figure S1A) (column 24) were added using a Biomek Liquid handler. Next, 10 µL of 53 µM ATP stock solution was added to each well with a different multiplex dispenser (Biotech Inc.) to a final ATP concentration of 21 μM. The plates were centrifuged for 1 minute at 1000 rpm then incubated for 2 hours at room temperature with shaking. Then, 15 µL of Kinase-Glo (Promega Inc.) diluted 2:1 with solution of 55.33 mM HEPES, 55.33 mM MgCl₂, pH 7.4 was added to each well using a Multidrop dispenser. Plates were centrifuged again for 1 minute at 1000 rpm and incubated for 10 minutes under agitation at room temperature. Luminescence was read on an EnVision Multilabel plate reader (PerkinElmer, Inc.).