Inhibitors of DNA Polymerase Beta: Hit Validation in HIV-RT 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 b). Although our understanding of the dNMP incorporation reaction by human pol b includes crystal structures representing stages of the catalytic more ..
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 b). Although our understanding of the dNMP incorporation reaction by human pol b 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 b via qHTS and 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 b. Thus, we anticipate the pol b-specific probes emerging from this research will serve as starting points in the development of clinical agents for use in down regulation of DNA base excision repair during therapeutic interventions against cancer.
After the completion of the qHTS against the Molecular Libraries Small Molecular Repository (MLSMR), cherry picked compounds were confirmed in the primary qHTS pol beta assay and further characterized in several follow-up assays. To avoid compound promiscuity, we selected a completely unrelated viral polymerase to polymerase beta, HIV reverse transcriptase(RT) as a counter screen. There are distinct differences between the two polymerases, such as source of host and HIV RT is responsible for transcribing the virus' single stranded RNA into double-stranded DNA after entry into its host cell. Hence, if a compound were to inhibit both enzymes, it could be assumed that compounds probably inhibit wide class of DNA polymerases. To us, a favorable outcome in this assay is inactivity.
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 stepwise description of the 1536-well assay is shown in next Table. Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol beta in columns 1, 2, and 5-48) was dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). The plates were incubated for 15 min at room temperature, and 1 uL substrate (50 nM final concentration) were added to start the reaction and immediately read at 0 min read on Viewlux reader. The plate were further incubated for 10 min at room temperature, and then read on Viewlux again. Throughout the screen, reagent bottle and all liquid lines were chilled and made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a double-dipping step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the beginning and the end of each library in order to monitor for and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity overtime. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts). A four parameter Hill equation was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)