Implementation of a 220,000-compound HCS campaign to identify disruptors of the interaction between p53 and hDM2 and characterization of the confirmed hits

Journal of Biomolecular Screening
Drew D DudgeonPaul A Johnston

Abstract

In recent years, advances in structure-based drug design and the development of an impressive variety of high-throughput screening (HTS) assay formats have yielded an expanding list of protein-protein interaction inhibitors. Despite these advances, protein-protein interaction targets are still widely considered difficult to disrupt with small molecules. The authors present here the results from screening 220,017 compounds from the National Institute of Health's small-molecule library in a novel p53-hDM2 protein-protein interaction biosensor (PPIB) assay. The p53-hDM2 positional biosensor performed robustly and reproducibly throughout the high-content screening (HCS) campaign, and analysis of the multiparameter data from images of the 3 fluorescent channels enabled the authors to identify and eliminate compounds that were cytotoxic or fluorescent artifacts. The HCS campaign yielded 3 structurally related methylbenzo-naphthyridin-5-amine (MBNA) hits with IC(50)s between 30 and 50 microM in the p53-hDM2 PPIB. In HCT116 cells with wild-type (WT) p53, the MBNAs enhanced p53 protein levels, increased the expression of p53 target genes, caused a cell cycle arrest in G1, induced apoptosis, and inhibited cell proliferation with an IC(50...Continue Reading

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Citations

Oct 27, 2014·Carcinogenesis·Lucia Morgado-PalacinManuel Serrano
May 21, 2013·Current Opinion in Chemical Biology·Samuel A Hasson, James Inglese
Sep 1, 2015·Assay and Drug Development Technologies·Paul A JohnstonJennifer R Grandis
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Sep 11, 2019·Assay and Drug Development Technologies·Ashley T FancherPaul A Johnston
Jan 1, 2011·MedChemComm·Kareem KhouryAlexander Dömling
Dec 31, 2020·International Journal of Molecular Sciences·Ekaterina S PotekhinaDmitry S Bilan

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Methods Mentioned

BETA
ubiquitination
xenograft
biosensor
interaction
immunoprecipitation
electrophoresis
fluorescence-activated cell sorting
FACS
flow cytometry
surface plasmon resonance

Software Mentioned

ArrayScan V
XLfit
ArrayScan
Biacore
WinMDI
GraphPad Prism
Leadscope Enterprise
ArrayScanV
Multigauge

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Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis