NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

NCGC Assay Overview:
The rate of false hits is dramatically reduced by the qHTS approach (Inglese et al, PNAS, 103, 1147 (2006)) as spurious high- or low-response wells are quickly revealed when plotted in the context of concentration response. Occasionally, the artifactual effect may originate from the compound#s own spectral or other biophysical properties which generally tend to track its assay concentration. Pre-screening/profiling the library with respect to the compounds# spectral properties should help one exclude false positives early on. While the most informative fluorescent profiling data set is obtained by performing a full excitation and emission scan of each well by using a monochromator-based plate reader, such an approach is practical only on a very limited scale as scanning each well requires long time. An alternative approach, applied here, is to perform plate reader scans using selected excitation and emission wavelength pairs to generate a multidimensional relational database that correlates compound concentration and the respective relative fluorescence intensity at a given wavelength set. A standard curve of rhodamine present on each plate as six-point concentration series, was used to compute a normalized fluorescence response, termed dye-equivalent concentration, for each compound at each concentration.

NCGC Assay Protocol Summary:

Buffer:
100 mM Tris buffer, pH 8.0

Dye standard plate:
The wells in columns 1 through 4 of a Kalypsys polypropylene 1536-well compound plate were filled with standard solutions of fluorophore corresponding to the spectral region of interest. Solutions of rhodamine were placed in the following wells: E1, F1: 3 mM, E2, F2: 300 uM, E3, F3: 30 uM, E4, F4: zero (DMSO), G1, H1: 3 uM, G2, H2: 900 nM, G3, H3: 300 nM, G4, H4: zero (DMSO).

Assay Steps:
Six uL of buffer were dispensed to 1536-well Greiner black plates. Compounds and dye standards (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using 547 nm and 598 nm excitation and emission filter set.

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, Rhodamine , Fluorescent Profiling.

Compound Classification and Ranking:

1. A log of maximum dye equivalent concentration was determined for each sample. Samples that were -9 or below in log of max dye equivalency were declared as inactives. Samples between -8 and -9 were inconclusive and samples greater than -8 were declared as active. An active is considered to be a sample that showed significant fluoresence at this particular dye's excitation and emission wavelengths.

2. For all inactive samples, PUBCHEM_ACTIVITY_SCORE is 0. For all active samples, ranking of dye equivalency was normalized from 100uM (score of 100) to 1nM (score of 0). A higher score is given to samples that exhibited more fluoresence in this assay.