Tumor suppressor plays a key role in suppression of tumorigenesis and is frequently inactivated, mainly by point mutation, during human carcinogenesis. In general, cancer cells with a p53 mutation are more resistant to current chemoradiation therapies. Our long range goal is to discover and develop a novel class of small molecule drugs, that reactivate p53 from its mutant form via conformational changes, as anticancer agents acting alone or in combination with chemoradiation. The objective of this application is to gain access to the HTS (High Throughput Screening) at the MLPCN (Molecular Libraries Probe Production Centers Network) to identify and characterize small molecules that induce conformational changes in mutant p53, leading to its reactivation. Our central hypothesis is that structural conformation of a mutant p53 can be changed to wild type by a small molecule, leading to reactivation of p53 to suppress growth of cancer cells by inducing growth arrest and apoptosis.
Specific aims are 1) to identify small molecules, via screening 300,000 compounds at the MLSMR (Molecular Libraries Small Molecule Repository), that reactivate p53 through conformational change using a luciferase reporter driven by a wt p53 responsive element and 2) to conduct secondary confirmation and counterscreening to filter out potential false positives, followed by biochemical validation for p53 reactivation and biological validation for cancer cell killing as single agent or in combination with radiation. The screening assay was developed in our laboratory in a 96-well cell-based luciferase reporter assay and has been miniaturized into a 1536-well plate format. This luciferase-reporter-based assay uses p53-null H1299 lung cancer cells which have been stably co-transfected with a temperature sensitive p53 mutant, p53-A138V (analine-to-valine mutation at codon 138), and a p53-responsive BP100-luciferase reporter, driven by intron-1 fragment of the Mdm2 gene (designated as H1299-p53ts-Luc). The compound that changes p53 conformation from mutant to wild type will result in transactivation of a wt p53-dependent reporter for increased luciferase activity. The assay window can be as high as 60-fold. Initial screening of the LOPAC library identified four nocodazole series of compounds and three were confirmed after a counterscreening. Upon successful completion of this proposed study, we expect to identify and validate at least one novel class of small molecules that reactivate p53 via inducing conformational change of p53 mutants. These molecules would have the potential to be further developed, after SAR (Struction Activity Relationsip) optimization, for clinic use as a single agent as well as in combination with chemoradiation for effective treatment of human cancer harboring a mutant p53.

Public Health Relevance

Tumor suppressor is mutated in 50% human cancers and human cancers with p53 mutation are in general more resistant to chemo-radiation therapy. The goal of this proposal is to identify small molecule compounds that are able to restore p53 function. These compounds would likely have the potential to be further developed as anticancer agents alone or in combination with chemoradiation.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-BST-J (50))
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Charya, Ananth V
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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