Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens

Abstract

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi. We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen

“…The Rh-PBP assay measures the WIP1-catalyzed release of inorganic phosphate from the peptide substrate. 47 The Rh-PBP and RapidFire MS assays were in excellent agreement with 172 of 195 (88%) cherry-picks showing activity in both. An Amplex Red counter assay was then performed in the presence of reducing agents according to previously published methods.…”

“…The RapidFire MS assay uses a physiologically relevant phosphorylated P53 peptide substrate and quantifies the dephosphorylated reaction product. 47 The reported assay used GSK2830371 as a control, which showed an IC 50 value of 3.5 nM, and demonstrated the ability to identify inhibitors of WIP1 phosphatase from a pilot screen (Figure 1A). Additional efforts were required to validate the use of this assay for discovering small-molecule WIP1 activators.…”

“…47 Expression and purification of the WIP1 enzyme (1−240), the wild-type PPM1A enzyme, and the phosphate-binding protein (PBP), as well as the rhodamine-PBP conjugate synthesis, were performed as described previously. 47,53 RapidFire MS and Rhodamine-Labeled PBP (Rh-PBP) WIP1 Assays. The RapidFire MS and rhodamine-labeled PBP (Rh-PBP) WIP1 assays were developed, optimized, and validated as previously reported.…”

“…The RapidFire MS and rhodamine-labeled PBP (Rh-PBP) WIP1 assays were developed, optimized, and validated as previously reported. 47 Assay protocols are provided in Tables S1 and S2, respectively. For the RapidFire MS assay, the high-speed solid-phase extraction (SPE) was performed with an Agilent RapidFire 365 system with a C4 Type A SPE cartridge and three HPLC pumps, which injected the analytes into a coupled Agilent 6470 triple quadrupole mass spectrometer (Agilent, Santa Clara, CA) equipped with an AJS ESI interface source tuned and calibrated in positive mode.…”

“…Flow rates of solvent for loading, washing, and elution, and quadrupoles resolution, voltage, gas flow, and temperature were described previously. 47 As an internal standard for normalization and reaction product quantification, 1 μM of heavy-labeled product peptide The quality of the primary assay was assessed by calculating the Z′ factor, 54 using the formula where σ p is the standard deviation of the positive controls, σ n is the standard deviation of the negative controls, μ p is the mean of the positive controls, and μ n is the mean of the negative controls.…”