Small-molecule inhibitor ELISA screen

We obtained the GlaxoSmithKline published kinase inhibitor set, PKIS (Dranchak et al., 2013) to evaluate potential SPAK drug inhibitor candidates. The pilot inhibitor screen tested the capability of 320 compounds to inhibit the kinase activity of purified co-expressed S^P382 WNK1 (1-661)/ WT SPAK using an ELISA based screen monitoring a reduction in NKCC1 phosphorylation with the NKCC1 phospho-specific antibody, T^PNKCC1. 10 nL of each inhibitor compound (final concentration 20 μM) or DMSO vehicle controls were added to dry 384 well-flat bottom low volume non-binding assay plates (Corning) using an Echo 550 Acoustic dispenser (Labcyte). 125 nM SPAK (from the co-expressed variant), and 100 nM GST-NKCC1 were mixed with Kinase reaction buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 20 mM MgCl2, 2 mM DTT, 0.1% BSA) that did not contain ATP, and 4 μL of the mixture was distributed into each well of the assay plate. The assay plates were centrifuged at 200 g for 1 min. then incubated at RT for 30 min. Following inhibitor incubation, the kinase reaction was initiated with 1 μL of 50 μM ATP (final concentration 10 uM) or 1 uL of Kinase reaction buffer for negative controls and reacted for 1 hr at 37°C. The reaction was then quenched with 5 μL Kinase Stop buffer (100 mM Tris pH 8, 300 mM NaCl, 40 mM EDTA) and incubated for 1 min. with shaking at 450 rpm. 2 uL of each quenched reaction was added to 18 μL TBS (50 mM Tris pH 8, 150 mM NaCl) in white 384-well Nunc Maxisorp plates (Thermo Scientific) using a PlateMate Plus (Thermo Scientific). The plate was shaken for 1 min. at 450 rpm, sealed and incubated at 4°C overnight to allow the protein contents of the reaction to bind to the Maxisorp plate. The next morning the solution was removed, and the plates were then subjected to 3 washes in TBST (50 mM Tris pH 7.5, 150 mM NaCl. 0.05% Tween 20) for 5 min each wash with shaking at 400 rpm. The plates were blocked with TBST buffer containing 3% BSA (w/v) at RT for 2 hr at RT. The blocking buffer was removed, and the plates were immunoblotted with 1:50 000 dilution of T^PNKCC1 in blocking buffer for 2 hr at RT. The plates were then subjected to 3 washes in TBST for 5 min each wash with shaking at 400 rpm, followed by incubating with 1:10 000 dilution of 2° DAR-HPR antibody in blocking buffer for 1 hr at RT. The plates were washed again 3 times in TBST for 5 min each wash with shaking at 400 rpm. Signal was detected by SuperSignal ELISA Pico Chemiluminescent substrate (Thermo Scientific) and read on an EnVision multi-label plate reader (Perkin-Elmer).