AID	Name	Type	Comment
2386	Probe Development Summary for Inhibitors of Bloom's syndrome helicase (BLM)	summary	Probe Development Summary for Inhibitors of Bloom's syndrome helicase (BLM)
2585	qHTS Confirmation Assay for Inhibitors of Bloom's syndrome helicase (BLM)	confirmatory	qHTS Confirmation Assay for Inhibitors of Bloom's syndrome helicase (BLM)
2528	qHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)	confirmatory	qHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)
2364	qHTS Validation Assay for Inhibitors of Bloom's syndrome helicase (BLM)	confirmatory	qHTS Validation Assay for Inhibitors of Bloom's syndrome helicase (BLM)
2712	Counterscreen for BLMA Inhibitors: ADP Fluorescence Polarization Displacement Assay	confirmatory	Counterscreen for BLMA Inhibitors: ADP Fluorescence Polarization Displacement Assay

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 DNA unwinding assay measured by gel electrophoresis (32P-labeled substrate) 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.

Initial screening hits were validated by the standard assay for measuring DNA helicase activity, which utilizes gel electrophoresis to monitor the helicase-mediated unwinding of a radiolabeled DNA substrate.

Unwinding assays using BLM helicase was carried out in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 2 mM ATP, 1 mM DTT, and 0.1 mg/ml BSA. Optimal substrate and enzyme concentrations as well as reaction times were determined experimentally. All reactions were performed at 37 degrees C and terminated by the addition of loading buffer containing 20 mM EDTA and 2.5% bromophenol blue. Reaction products were separated on 15% nondenaturing polyacrylamide gels in 1x TBE at 30 mA for 1 hr at 4 degrees C. The gels were then dried and visualized by autoradiography (Kodak BioMax MR-1).

Keywords, BLMA, RecQ Helicase, RecQ-like type 2, Bloom syndrome protein

Compounds that were inactive were given a PUBCHEM_ACTIVITY_SCORE of 0. Active compounds got a PUBCHEM_ACTIVITY_SCORE of -10*Log_IC50 rounded to the closest whole number.

Grant Number: MH087284-01