qHTS for Inhibitors of WRN Helicase: BLM Helicase Counterscreen for WRN Inhibitors
Inhibition of DNA repair is proposed as a strategy for combating cancer. Synthetic lethality is an approach that exploits preexisting DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated DNA repair deficiency. Because helicases play critical roles in the DNA damage response and in DNA repair, particularly in actively more ..
BioActive Compounds: 368
Inhibition of DNA repair is proposed as a strategy for combating cancer. Synthetic lethality is an approach that exploits preexisting DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated DNA repair deficiency. Because helicases play critical roles in the DNA damage response and in DNA repair, particularly in actively dividing and replicating cells, characterization of synthetic lethal relationships of DNA helicases may be of value in developing improved anticancer treatment strategies; moreover, small molecules that specifically target a given DNA helicase may be useful for understanding its role in cellular nucleic acid metabolism. The goal of this project is to identify small molecule, non-covalent chemical inhibitors of the Werner syndrome (WS) helicase (WRN), which plays an important role in cell proliferation, the replication stress response, and DNA repair. Werner syndrome is a premature aging disorder that displays many clinical symptoms of aging at an accelerated rate. The WRN gene product that is defective in the chromosomal instability disorder has DNA helicase and exonuclease activities and interacts with a number of nuclear proteins to maintain genomic stability.
A 1536-well format, fluorescence based, high-throughput screen (AID 651768) has been developed in collaboration with the Brosh Laboratory at the National Institute of Aging (NIA) to screen for inhibitors of the WRN. This counterscreen is used to determine the activity of the validated WRN inhibitors against another related helicase, the Bloom's syndrome (BLM) helicase. Compounds / series that exhibit a 10-fold selectivity will be will be included for further consideration and target characterization of the WRN inhibitors.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH096530
Assay Submitter (PI): Robert Brosh, National Institute of Aging
Three uL of reaction buffer (25 mM Tris-HCl pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.01% Tween-20, and 1 nM BLM) was dispensed into a 1536-well Greiner black assay plate via solenoid-valve based nanoliter dispensers. Compounds (23 nl each in columns 5-48) and control (23 nl each in column 2 as dose-response) were transferred to the assay plate via a Kalypsys pin-tool equipped with a 1536-pin array. The plates were incubated for 15 min at room temperature, followed by the addition of 1 ul substrates (100 nM DNA and 2 mM ATP, final concentrations) to start the reaction. The plates were transferred into a ViewLux CCD imager where the reaction was measured in a discontinuous kinetic mode (two reads spaced 15 minutes apart, with intermediate room temperature incubation of the covered assay plate) using the standard red-fluorescence optics (excitation filter 525 nm, emission filter 598 nm). Enzyme-free and vehicle (DMSO) controls were included for signal normalization.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)