Hazuda D, Felock P J, Hastings J C, Pramanik B, Wolfe A L
Department of Antiviral Research, Merck Research Laboratories, West Point, PA 19446, USA.
Drug Des Discov. 1997 May;15(1):17-24.
An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.
逆转录病毒复制过程中的一个关键步骤是将病毒基因组的DNA拷贝整合到宿主细胞的基因组中。整合过程包括一系列由病毒酶整合酶催化的有序核酸内切和DNA链转移反应。在细胞培养中HIV-1增殖需要整合酶活性，这使得该酶成为化疗干预的潜在靶点。因此，我们开发了一种非放射性微孔板检测方法，可用于从随机化学筛选中鉴定整合酶的新型抑制剂，并用于从天然产物中通过生物测定法分离抑制剂。该检测方法将反应途径中的各个步骤解偶联，因此可用于表征抑制剂。在本专著中，我们以一系列先前描述的整合酶抑制剂为例，描述了该方法的一系列改进，这些改进有助于进行此类机制研究。
