Document Detail

Implementation of a 220,000-compound HCS campaign to identify disruptors of the interaction between p53 and hDM2 and characterization of the confirmed hits.
MedLine Citation:
PMID:  20639499     Owner:  NLM     Status:  MEDLINE    
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) ~4 microM. The prototype disruptor of p53-hDM2 interactions Nutlin-3 was more potent than the MBNAs in the p53-hDM2 PPIB assay but produced equivalent biological results in HCT116 cells WT for p53. Unlike Nutlin-3, however, MBNAs also increased the percentage of apoptosis in p53 null cells and exhibited similar potencies for growth inhibition in isogenic cell lines null for p53 or p21. Neither the MBNAs nor Nutin-3 caused cell cycle arrest in p53 null HCT116 cells. Despite the relatively modest size of the screening library, the combination of a novel p53-hDM2 PPIB assay together with an automated imaging HCS platform and image analysis methods enabled the discovery of a novel chemotype series that disrupts p53-hDM2 interactions in cells.
Drew D Dudgeon; Sunita Shinde; Yun Hua; Tong Ying Shun; John S Lazo; Christopher J Strock; Kenneth A Giuliano; D Lansing Taylor; Patricia A Johnston; Paul A Johnston
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2010-07-16
Journal Detail:
Title:  Journal of biomolecular screening     Volume:  15     ISSN:  1552-454X     ISO Abbreviation:  J Biomol Screen     Publication Date:  2010 Aug 
Date Detail:
Created Date:  2010-08-23     Completed Date:  2010-12-01     Revised Date:  2011-05-23    
Medline Journal Info:
Nlm Unique ID:  9612112     Medline TA:  J Biomol Screen     Country:  United States    
Other Details:
Languages:  eng     Pagination:  766-82     Citation Subset:  IM    
University of Pittsburgh Drug Discovery Institute, School of Medicine, Pittsburgh, PA, USA.
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MeSH Terms
Apoptosis / drug effects
Biosensing Techniques
Cell Cycle / drug effects
Cell Line, Tumor
Cell Proliferation / drug effects
DNA Damage
High-Throughput Screening Assays / methods*
Naphthyridines / pharmacology
Protein Binding / drug effects
Proto-Oncogene Proteins c-mdm2 / metabolism*
Reproducibility of Results
Small Molecule Libraries / analysis*,  chemistry,  pharmacology*
Tumor Suppressor Protein p53 / metabolism*
Grant Support
Reg. No./Substance:
0/Naphthyridines; 0/Small Molecule Libraries; 0/Tumor Suppressor Protein p53; EC protein, human; EC Proteins c-mdm2

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