| Assay Method Information | |
| | Factor XIa 1536 HTS Dose Response Confirmation |
| Description: | Factor XIa (0.23 ug/mL) was incubated with Boc-Glu-Ala-Arg-AMC substrate (15 uM) in 10 uL of assay buffer for 2 hr at room temperature. Read fluorescence (excitation 355, emission 460) on Envision reader. HTS hits were confirmed on single compounds by IC50 determination. IC50 plates contained compounds in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 1.5 nM. 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 using the following equation: % Activity = 100*((signal-blank mean)/(control mean-blank mean)) 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. 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. Activity outcome is reported as follows: (1) IC50 <50 uM in all three IC50 determinations = active (2) IC50 <50 uM in only 1 or 2 out of 3 determinations = inconclusive (3) IC50 >50 uM, percent inhibition 30-50% at 50 uM = inconclusive (4) IC50 >50 uM, percent inhibition <30% at 50 uM = inactive |
| Affinity data for this assay | |
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