Pharmacology of HIV Viral DNA & Retroviral Integrases

为了进一步扩大开发抗逆转录病毒药物的靶点数量，我们正在使用重组酶和对应于前病毒末端的短寡核苷酸（LTR）进行体外测定，研究HIV-1整合酶抑制剂。整合酶是抑制剂开发的基本靶点，因为它对病毒复制至关重要。它由病毒基因组编码，没有细胞等价物。我们的实验室开创了这一研究领域，并报告了各种抑制剂家族。我们已经就这一主题写了几篇评论;最近的一篇将于2004年初发表。今年，第一类HIV-1整合酶抑制剂已进入临床试验。我们正在与Terrence Burke博士（药物化学实验室，CCR，NCI）和Vinay Pathak博士（抗病毒药物耐药性计划，CCR，NCI）合作研究这些二酮酸（DKA）衍生物。我们已经阐明了这种新型化合物的结构-活性关系，并发现DKA区分野生型和突变型HIV-1整合酶以及整合酶的两种金属辅因子镁和锰。我们的目标是阐明酶-DNA复合物中的药物结合位点，并发现具有更大治疗指数和/或新结构基序的药物。我们发现二酮酸的叠氮衍生物是有效的和选择性的抗整合酶抑制剂，并且是抗病毒的。叠氮基可以螯合酶催化位点中的二价金属。这些衍生物已申请专利。我们还在研究新型抑制剂，它们可以通过与前病毒DNA末端结合来阻止整合，以及小肽和核苷酸抑制剂。肽抑制剂也是抗病毒的，并已获得专利。我们也正在研究整合酶和它的DNA底物之间的分子相互作用，使用酶-DNA交联试验，以模拟药物-酶-DNA相互作用。一项使用苯并[a]芘二醇环氧化物dG-N2加合物进行小沟扫描的研究正在《生物化学杂志》上发表。我们最近发现了一种病毒DNA碱基与酶之间的新型相互作用。

In an effort to further extend the number of targets for development of anti-retroviral agents, we are studying HIV-1 integrase inhibitors using in vitro assays using recombinant enzyme and short oligonucleotides corresponding to the proviral ends (LTR's). Integrase is a rationale target for inhibitor development because it is essential for viral replication. It is encoded by the viral genome and does not have a cellular equivalent. Our laboratory has pioneered this research field and reported various families of inhibitors. We have written several reviews on this topic; the most recent will be published in early 2004. This year, the first class of HIV-1 integrase inhibitors has been introduced in clinical trials. We are investigating these diketo acid (DKA) derivatives in collaboration with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, CCR, NCI) and Dr. Vinay Pathak (Antiviral Drug Resistance Program, CCR, NCI). We have elucidated the structure-activity relationship for this new type of compounds, and found that DKAs discriminate between wild-type and mutant HIV-1 integrase and between magnesium and manganese, the two metal cofactors for integrase. Our goal is to elucidate the drug binding site(s) in the enzyme-DNA complex, and to discover agents with a greater therapeutic index and/or novel structural motifs. We discovered that azido derivatives of diketo acids are potent and selective anti-integrase inhibitors, and are antiviral. The azido group can chelate the divalent metal in the enzyme catalytic site. A patent application has been filed for these derivatives. We are also studying novel types of inhibitors that can prevent integration by binding to the proviral DNA ends as well as small peptide and nucleotide inhibitors. The peptide inhibitors are also antiviral and have been patented. We are also studying the molecular interactions between integrase and its DNA substrate using enzyme-DNA crosslinking assays in order to model drug-enzyme-DNA interactions. A study using minor groove scanning with benzo[a]pyrene diol epoxide dG-N2 adduct is in press in The Journal of Biological Chemistry. We recently found a novel interaction between one of the viral DNA bases and the enzyme.