Biosensor analysis of drug-target interactions:: Direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors

The sensitivity of BIACORE technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. The technology requires no labeling and provides information on the stability of the compound/target complex with a single injection of the compound. This is useful for qualifying hits obtained in a primary screen and in lead optimization. Although immobilized targets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds may exhibit broad protein selectivity rather than target specificity. A reliable direct binding assay for compounds binding to immobilized thrombin using a combination of two reference surfaces, a dextran surface for subtraction and calibration of solvent effects and a protein surface for identification of compounds that tend to bind proteins, has been developed. Eleven compounds with known binding specficity to thrombin and 159 additional compounds were investigated. All compounds with known binding specificity were identified at 1 and 10 mu M concentration. One additional compound was scored as positive. The direct binding assay compared favorably with two competitive assay formats, a surface competitive assay and a inhibitor in solution assay, that were examined in parallel. (C) 2000 Academic Press.