AID	Name	Type	Comment
2367	Probe Development Summary for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)	summary	Probe Development Summary for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)
2353	qHTS Validation Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)	confirmatory	qHTS Validation Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)
593	qHTS Assay for Spectroscopic Profiling in Fluorescein Spectral Region	other	Fluorescein region spectral profiling screen
594	qHTS Assay for Spectroscopic Profiling in Rhodamine Spectral Region	confirmatory	Rhodamine region spectral profiling screen
2549	qHTS Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)	confirmatory	qHTS Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)

In order to gain further insight into the mode of action of the RECQ1 screening hits, we have profiled them in a set of miniaturized fluorescence polarization assays designed to report on compounds which competitively displace either co-substrate (ATP or DNA). The appropriate fluorescently-labeled probe was used: BODIPY Texas Red-labeled ADP was expected to be competed off by ATP-competitive inhibitors, while single-TAMRA labeled forked-duplex or short single-stranded DNA molecules served as probes for DNA-competitive compounds.

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), containing 300 nM RECQ1 and 500 nM BODIPY Texas Red-ADP (Invitrogen) were dispensed into a 1536-well Greiner black assay plate via solenoid-valve based nanoliter dispensers; 500 nM BODIPY Texas Red-ADP without RECQ1 protein served as a fully-displaced control. Compounds (23 nl each in columns 5-48) were transferred via a Kalypsys pintool equipped with a 1536-pin array. The plates were incubated for 15 min at room temperature and were then transferred into a ViewLux high-throughput CCD imager, where the endpoint fluorescence polarization was measured using 525 nm excitation and 598 nm emission protocol.

Compound Ranking:

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.

Grant Number: MH087284-01