qHTS Validation Assay for Inhibitors of Bloom's syndrome helicase (BLM)
Survival of cells and the faithful propagation of the genome depend on elaborate mechanisms of detecting and repairing DNA damage. Treatment of advanced cancer relies on radiation therapy or chemotherapy, which kill cancer cells by causing extensive DNA damage. It is often found, that cancer cells develop resistance to therapy through enhanced activity of DNA repair functions; this has led to an increased interest in developing drugs that interfere with DNA repair, which could sensitize cancer cells to conventional therapy. ..more
BioActive Compounds: 30
Survival of cells and the faithful propagation of the genome depend on elaborate mechanisms of detecting and repairing DNA damage. Treatment of advanced cancer relies on radiation therapy or chemotherapy, which kill cancer cells by causing extensive DNA damage. It is often found, that cancer cells develop resistance to therapy through enhanced activity of DNA repair functions; this has led to an increased interest in developing drugs that interfere with DNA repair, which could sensitize cancer cells to conventional therapy.
This validation qHTS assay pertains to human BLM, which is important in resolving abnormal DNA structures formed during replication or homologous recombination. Shutting down the expression of BLM leads to chromosomal instability and higher radiation sensitivity in cultured cells.
The validation assay was a fluorescence quenching based kinetic qHTS for BLM Helicase DNA unwinding. The activity was measured as ATP-dependent separation of a 20-bp DNA duplex extended by 30-nt single-stranded tails (forked duplex). The forked DNA substrate was tagged with rhodamine fluorophore (carboxytetramethyl rhodamine, TAMRA) and BHQ-2 (Black Hole Quencher 2) dark quencher. Strand separation results in an increase in the fluorescence of TAMRA (excitation 525 nm, emission 598 nm). This substrate construct operates in a red-shifted region where very few compound library members have been noted to fluoresce (PubChem AIDs 593 and 594). An additional feature of the assay is the inclusion of 2.5 ug/ml poly (dIdC), to reduce interference by compounds such as DNA intercalators, a major source of false inhibitors.
Ian Hickson, University of Oxford
Opher Gileadi, Structural Genomics Consortium, University of Oxford
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Three uL of reaction buffer (25 mM Tris-HCl (pH 8.0), 5 mM NaCl, 2 mM MgCl2, 1mM DTT, 0.01% Tween- 20, and 2.5 ug/ml poly(dI-dC), including enzyme (50 nM BLM), were 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 via a Kalypsys pintool equipped with a 1536-pin array. The plates were incubated for 15 min at room temperature, and then 1 ul of substrates (200 nM DNA and 2 mM ATP, final concentrations) was added to start the reaction. The plate was transferred into a ViewLux high-throughput CCD imager where the reaction progress was measured in a discontinuous kinetic mode (two reads spaced 2 hours apart, with intermediate room temperature incubation of the covered assay plate) by using standard red-fluorescence optics (excitation filter 525 nm, emission filter 598 nm). Enzyme-free and vehicle (DMSO) controls were included for signal normalization.
Keywords, BLMA, RecQ Helicase, RecQ-like type 2, Bloom syndrome protein
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)