Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.
Victor Clausse
Dingyin Tao
Subrata Debnath
Yuhong Fang
Harichandra D Tagad
Yuhong Wang
Hongmao Sun
Christopher A LeClair
Sharlyn J Mazur
Kelly Lane
Zhen-Dan Shi
Olga Vasalatiy
Rebecca Eells
Lynn K Baker
Mark J Henderson
Martin R Webb
Min Shen
Matthew D Hall
Ettore Appella
Daniel H Appella
Nathan P Coussens
10779/crick.11619438.v1
https://crick.figshare.com/articles/journal_contribution/Physiologically_relevant_orthogonal_assays_for_the_discovery_of_small-molecule_modulators_of_WIP1_phosphatase_in_high-throughput_screens_/11619438
Wildtype 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, inorganic phosphate. 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.
2020-01-15 16:49:42
RapidFire
Wip1
assay
cancer
cancer therapy
enzyme kinetics
fluorescence
high-throughput screening (HTS)
kinetics
mass spectrometry (MS)
oncogene
phosphatase
phosphorylation
therapeutics
Webb FC001211
Biochemistry & Molecular Biology
06 Biological Sciences
11 Medical and Health Sciences
03 Chemical Sciences