Espeseth A S, Felock P, Wolfe A, Witmer M, Grobler J, Anthony N, Egbertson M, Melamed J Y, Young S, Hamill T, Cole J L, Hazuda D J
Departments of Antiviral Research, Medicinal Chemistry, and Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA.
Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11244-9. doi: 10.1073/pnas.200139397.
Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.
诸如L-731,988之类的二酮酸是HIV-1整合酶的强效抑制剂，可抑制细胞中的整合及病毒复制。这些化合物具有独特的能力，即在不影响3'末端加工的情况下抑制整合酶的链转移活性。为了理解这种独特抑制特性的原因，我们开发了一种闪烁邻近分析方法，该方法可用于分析放射性标记抑制剂的结合及整合酶功能。结果表明，L-731,988的高亲和力结合需要在HIV-1长末端重复序列上组装特定复合物。L-731,988与该复合物的相互作用以及其在链转移中的功效可被与靶底物的相互作用所消除，这表明抑制剂与靶DNA之间存在竞争。因此，L-731,988的结合位点和靶底物的结合位点与供体底物的不同，并且由整合酶仅在与病毒末端组装后才采用的一种构象所定义。这些结果阐明了二酮酸抑制链转移的基础，并对针对整合酶的HIV-1药物研发工作具有启示意义。
