| Assay Method Information | |
| | Factor XIIa Dose Response Confirmation from Single Well HTS |
| Description: | HTS was performed on a total of 33,068 compounds of the MLSCN library, 23,017 of which were not in the mixture HTS plates used previously. These compounds were plated as single components of 384 well plates at 0.5 mM stock concentration, and were diluted 50-fold into 10 ul 384 well assay plates (final concentration 10 uM each compound). The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of with Boc-Gln-Gly-Arg-AMC. Percent activity was calculated for each dilution of each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks. Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively). Compounds that gave percent inhibition >40 in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above. A zero in the IC50 field indicates that >50% enzyme activity remained at the maximum compound concentration tested (50 uM), so the IC50 lies outside the range of compound concentrations tested and can be inferred to be >50 uM. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM. |
| Affinity data for this assay | |
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