| Assay Method Information | |
| | Fluorometric PAI-1/uPA IC50 Plate Assay |
| Description: | To determine the efficacy of various synthesized compounds as PAI-1 inhibitors, a fluorometric plate assay was carried out to measure the half maximal inhibitory concentration (IC50) of these compounds on recombinant active human PAI-1 in vitro. An IC50 is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. Stated another way, IC50 represents the concentration of a drug that is required for 50% inhibition in vitro. The IC50 of various compounds was measured using a fluorometric plate assay. PAI-1 inhibitor compounds were dissolved in DMSO to a final concentration of (10-50 mM), depending upon solubility. Compounds were then diluted in physiologic buffer (40 mM HEPES, 100 mM NaCl, 0.05% Tween-20, pH7.4) containing 10% DMSO and a dilution series (from 0 to 1000 uM depending on solubility) was prepared. 80 μL of compound was added per well to a 96-well black, opaque microplate in duplicate. 10 μL of 20 nM Recombinant active human PAI-1 (Molecular Innovations) in physiologic buffer (40 mM HEPES, 100 mM NaCl, 0.05% Tween-20, 10% DMSO, pH 7.4), or physiologic buffer with 15 mg/mL, or physiologic buffer with 10% human plasma, was added and the mixture was agitated for 15 minutes at room temperature. 10 μL of 25 nM uPA (Rheotromb ) was added to each reaction well and the plate was agitated for an additional 30 minutes at room temperature. Tripeptide aminomethylcoumarin Gly-Gly-Arg-AMC (Calbiochem) fluorogenic substrate (100 μL of 100 μM) was then added and residual uPA activity was determined based upon cleavage of this substrate. The rate of AMC release by uPA (fluorescence) was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. Controls included PAI-1 and uPA in the absence of compound and uPA alone. Percent PAI-1 inhibition was calculated using the following formula: [(uPA alone-uPA/PAI-1+ compound)/(uPA alone-PAI-1/uPA)]*100%. The IC50 was calculated using Graphit (IC50 0-100%). |
| Affinity data for this assay | |
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