qHTS for Inhibitors of WRN Helicase
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: 1678
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.
We will employ an optimized high-throughput screening assay that utilizes a fluorescence intensity modulation scheme with a fluorophore and quencher positioned near one another within a duplex deoxyoligonucleotide that contains a single forked structure. After a high-throughput screen of the ~350,000-member Molecular Libraries Small Molecule Repository (MLSMR) collection, followed by data analysis and hit selection, a panel of confirmatory, as well as secondary and ternary, assays will be employed to identify high-confidence WRN inhibitors. Structure-activity relationships will be investigated for the top candidate inhibitor series through medchem expansion. These are the results of the primary screening campaign against the MLSMR.
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 muL of reagents (buffer in columns 3 and 4 as negative controls; WRN at 20 nM final concentration in columns 1 and 2 as positive controls; columns 5-48 as test reactions) will be dispensed into a 1536-well Greiner black 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) will be transferred via a Kalypsys pintool equipped with a 1536-pin array. Plates will be incubated for 15 min at room temperature, and then 1 muL of substrate (100 nM forked duplex oligonucleotide and 2 mM ATP final concentrations) will be added to start reaction, and plate will be transferred into a ViewLux high-throughput CCD imager where reaction progress is measured in kinetic mode using standard red-fluorescence optics (excitation filter 525 nm, emission filter 598 nm).
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)