| Assay Method Information | |
| | Thrombin 1536 HTS Dose Response Confirmation |
| Description: | HTS was performed using 217,350 compounds of the MLSCN library individually plated into 10ul 1536 compound plates at a concentration of 2.5 mM each, which were diluted 500-fold into 5 ul 1536 well assay plates (final concentration 5 uM each compound). This assay was performed as a counterscreen to compare with a separate HTS campaign to isolate inhibitors of the strong protein-protein interactions among fibrin monomers responsible for fibrin gel formation. Since any inhibitors of the proteolytic activity of alpha-thrombin would inhibit an assay to detect fibrin formation, this counterscreen will identify direct alpha-thrombin inhibitors. The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of Boc-Val-Pro-Arg-AMC. alpha-thrombin (5.5 ng/mL) was incubated with Boc-Val-Pro-Arg-AMC substrate (15 uM) in 5 uL of assay buffer for 30 min at room temperature. 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. 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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