Inhibitors of DNA Polymerase Beta: Hit Validation in Radiolabeled Extension Assay
The base excision repair system in human cells is a target for therapeutic modulation of the response to irradiation treatment and DNA-damaging drugs. A key enzyme in this system is the bi-functional DNA polymerase known as DNA polymerase Beta (Pol Beta). Although our understanding of the dNMP incorporation reaction by human Pol Beta includes crystal structures representing stages of the more ..
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The base excision repair system in human cells is a target for therapeutic modulation of the response to irradiation treatment and DNA-damaging drugs. A key enzyme in this system is the bi-functional DNA polymerase known as DNA polymerase Beta (Pol Beta). Although our understanding of the dNMP incorporation reaction by human Pol Beta includes crystal structures representing stages of the catalytic cycle, research with small molecule probes or inhibitors to date has failed to provide the information necessary to effectively target this enzyme. Using an integrated approach involving discovery of small molecule probes for Pol Beta via HTS and medchem optimization in collaboration with the NIH Chemical Genomics Center, validation by crystallography and kinetics along with cell-based validation experiments, we will test the hypothesis that base excision repair can be strategically modulated in human cells by targeting Pol Beta. Thus, we anticipate the Pol Beta-specific probes emerging from this research will serve as starting points in the development of clinical agents for use in downregulation of DNA base excision repair during therapeutic interventions against cancer.
This assay is an orthogonal follow-up assay that looks at radiolabeled primer extension and was carried out by the assay provider.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH090863-01
Assay Submitter (PI): Samuel Wilson, National Institute of Environmental Health Sciences (NIEHS), NIH
A primer oligonucleotide is 5'-32P end-labeled using T4 polynucleotide kinase and 32P-[ATP]. This strand is then annealed to an unlabeled, undamaged complementary strand. 10mul reaction mixtures will be set up essentially as described for the strand displacement DNA synthesis assay utilizing 5nM of radiolabeled primer/template, 10nM of DNA polymerase in the corresponding optimized reaction buffer and various concentrations of the proposed inhibitor. Reactions are started by the addition of 100muM dTTP and incubated at 27 degrees C for 30 minutes. Replication products will be separated on 12%/8M urea polyacrylamide gels and visualized by PhosphorImager analysis. Percent extension of substrate to product is measured using standard, quantitative phosphoimager analysis.
PUBCHEM_ACTIVITY_OUTCOME is defined that if activity at 10 uM is 30% or above, substance is found to be active. If percent activity at 10 uM is below 30%, substance is found inactive.
PUBCHEM_ACTIVITY_SCORE is the closest whole number.
Data Table (Concise)