Novartis Institute for Tropical Diseases, Chromos 138670, Singapore.
Anal Biochem. 2009 Dec 15;395(2):195-204. doi: 10.1016/j.ab.2009.08.013. Epub 2009 Aug 13.
In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.
在药物发现中，假阳性的出现是寻找可开发成药物的先导化合物的主要障碍。在筛选活动中发现了一种能在低微摩尔水平抑制登革热病毒蛋白酶的小分子化合物。通过等温滴定量热法（ITC）和核磁共振（NMR）证实了与酶的结合。然而，随后进行的构效关系研究并没有产生更有效的先导化合物。为了进一步表征母体化合物及其类似物，我们开发了一种高速、低成本、定量荧光猝灭测定法。我们观察到特定的类似物猝灭登革热蛋白酶的荧光，并显示出 IC（50）值的变化。相比之下，非特异性结合的化合物不能猝灭其荧光，并且具有相似的 IC（50）值和陡峭的剂量反应曲线。我们使用单个 Trp-to-Ala 蛋白酶突变体和竞争性蛋白酶抑制剂 aprotinin 验证了该测定法。在结合测定中检测到的特定化合物进一步通过竞争性 ITC、NMR 和表面等离子体共振进行分析，并讨论了该测定法与这些生物物理方法相比的实用性。该测定法的灵敏度使其在药物发现中的命中发现和验证中非常有用。此外，该技术可以很容易地适应于研究其他蛋白质-配体相互作用。
