Thrombin Inhibition by Benzofuran Derivatives

Direct inhibition of thrombin by sulfated benzofuran derivatives was measured through a chromogenic substrate hydrolysis assay.^{18 (link),19 (link)} The buffer used in these experiments was 20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl₂, and 0.1% polyethylene glycol (PEG) 8000. Benzofuran derivatives (2 to 30 μL) at concentrations ranging from 4 μg/ml to 5 mg/ml were diluted with appropriate volume of assay buffer in PEG 20,000-coated acrylic cuvettes at 25 °C. To this solution was added 5 μL of thrombin solution to give approximately 5 nM initial thrombin concentration. After 10 min of incubation, 20 μL of 1 mM Spectrozyme TH was rapidly added and the residual thrombin activity was measured from the initial rate of increase in absorbance at 405 nm. Relative residual thrombin activity at each concentration of the inhibitor was calculated from the ratio of thrombin activity in the presence and absence of inhibitor. Logistic equation 1 was used to fit the dose-dependence of residual proteinase activity to obtain the IC₅₀ and the efficacy ΔY (= Y_M – Y₀) of inhibition.
In this equation, Y is the ratio of residual thrombin activity in the presence of inhibitor to that in its absence (fractional residual activity), Y_M and Y_O are the maximum and minimum possible values of the fractional residual proteinase activity, IC₅₀ is the concentration of the inhibitor that results in 50% inhibition of enzyme activity, and HS is the Hill Slope, which was set constant at 1. A current version of SigmaPlot (SPSS, Inc. Chicago, IL) was used to perform non-linear curve fitting in which Y_M, Y_O and IC₅₀ were allowed to float.