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

The sensitivity of BIACORE technology is sufficient for detection and chara cterization 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 obtai ned in a primary screen and in lead optimization. Although immobilized targ ets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds ma y exhibit broad protein selectivity rather than target specificity. A relia ble direct binding assay for compounds binding to immobilized thrombin usin g a combination of two reference surfaces, a dextran surface for subtractio n and calibration of solvent effects and a protein surface for identificati on of compounds that tend to bind proteins, has been developed. Eleven comp ounds with known binding specficity to thrombin and 159 additional compound s were investigated. All compounds with known binding specificity were iden tified at 1 and 10 mu M concentration. One additional compound was scored a s positive. The direct binding assay compared favorably with two competitiv e assay formats, a surface competitive assay and a inhibitor in solution as say, that were examined in parallel. (C) 2000 Academic Press.