Biosensor analysis of drug-target interactions:: Direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors
Biosensor analysis of drug-target interactions:: Direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors
复制标题
DOI：
10.1006/abio.1999.4406
复制
发表时间：
2000-02-01
影响因子：
2.9
通讯作者：
Deinum, J
中科院分区：
文献类型：
--
作者：
Karlsson, R;Kullman-Magnusson, M;Deinum, J
BIACORE技术的灵敏度足以检测和表征涉及低分子量化合物及其固定化蛋白质靶标的结合事件。该技术不需要标记，并且提供了关于单次注射化合物的化合物/靶复合物的稳定性的信息。这对于在主屏幕和线索优化中获得的合格命中很有用。虽然固定的目标可以重复使用，表面可能会慢慢恶化，溶剂效应可以扭曲化合物注射过程中的结合水平，一些化合物可能会表现出广泛的蛋白质选择性，而不是目标特异性。已经开发了一种可靠的直接结合测定法，用于结合到固定化凝血酶的化合物，使用两个参考表面的组合，用于减去和校准溶剂效应的葡聚糖表面和用于鉴定倾向于结合蛋白质的化合物的蛋白质表面。研究了11种已知与凝血酶结合特异性的化合物和159种其他化合物。在1和10 μ M浓度下鉴定了具有已知结合特异性的所有化合物。另外一种化合物被评分为阳性。直接结合试验与两种竞争性试验形式（表面竞争性试验和溶液中的抑制剂试验）进行了平行检查。(C)北京大学出版社.
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.