Pharmacology of HIV Viral DNA and Retroviral Integrases

HIV 病毒 DNA 和逆转录病毒整合酶的药理学

• 批准号: 8157202
• 负责人: YVES POMMIER
• 金额: $ 60.78万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157202
• 关键词: HIV; Integrase; Pharmacology; viral DNA

Integrase is encoded by the Pol gene from the HIV provirus and can be expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors (Nature Rev Drug Discovery 2005; Current Topics in Medicinal Chemistry 2009; Viruses 2010) and patented some with the aim of therapeutic development. Our current studies are focused on the discovery of novel chemotype integrase inhibitors to overcome resistance to raltegravir and target novel site of integrase. In the past year we have reported novel chemotypes derived from Vpr peptides. These short peptides represent a novel kind of inhibitors. We have shown they could serve as therapeutics by the addition of a poly-arginine tail that confers cellular uptake and antiviral activity. They can also been used to build non-peptidic derivatives. We have also reported novel synthetic chemotypes affiliated with the diketo acid strand transfer inhibitors (phtalimide and quinolinonyl diketo acid derivatives) (collaboration with Dr. Terrence Burke, Laboratory of Medicinal Chemistry, CCR, NCI, and with Dr. Roberto DiSanto, University of Rome, Italy). We have developed a panel of recombinant integrase proteins bearing the mutations observed in patients that develop resistance to raltegravir and elvitegravir. We have compared for the first time raltegravir and elvitegravir and shown that both drugs are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity of raltegravir and elvitegravir against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed strand transfer inhibitors. It is consistent with our pharmacological hypothesis (TIPS 2005) that the strand transfer inhibitors trap the integrase-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3-processing of the viral DNA. We recently characterized in several publications the biochemical enzymatic activities and drug sensitivities of the integrase mutants that confer clinical drug resistance. Those same mutants are now available to test our novel inhibitors. We are expanding these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of integrase after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. Notably, we found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Having established the assays with a battery of recombinant mutant integrase, we are in a position to discover novel chemotypes to overcome raltegravir and elvitegravir resistance.

整合酶由 HIV 原病毒的 Pol 基因编码，可以表达为活性重组蛋白。我们的实验室开创了整合酶抑制剂研究领域（PNAS 1993），发现了几个先导抑制剂家族（Nature Rev Drug Discovery 2005；Current Topics in Medicinal Chemistry 2009；Viruses 2010），并为一些治疗开发申请了专利。我们目前的研究重点是发现新型化学型整合酶抑制剂，以克服对拉替拉韦的耐药性并靶向整合酶的新位点。在过去的一年中，我们报道了源自 Vpr 肽的新化学型。这些短肽代表了一种新型抑制剂。我们已经证明，它们可以通过添加聚精氨酸尾部来作为治疗剂，从而赋予细胞摄取和抗病毒活性。它们还可用于构建非肽衍生物。我们还报道了与二酮酸链转移抑制剂（邻苯二甲酰亚胺和喹啉基二酮酸衍生物）相关的新型合成化学型（与 CCR、NCI 药物化学实验室的 Terrence Burke 博士和罗马大学的 Roberto DiSanto 博士合作） ， 意大利）。我们开发了一组重组整合酶蛋白，其中含有在对拉替拉韦和艾维拉韦产生耐药性的患者中观察到的突变。我们首次比较了拉替拉韦和埃替拉韦，结果表明这两种药物对链转移反应具有高度选择性，而对 3-加工反应的效力低 100 倍以上，并且对整合酶介导的崩解反应几乎没有活性。拉替拉韦和埃替拉韦对链转移（整合酶介导的 3 个反应之一）的选择性活性证明了临床开发的链转移抑制剂具有非常高的特异性。这与我们的药理学假设 (TIPS 2005) 一致，即链转移抑制剂通过在病毒 DNA 3 次处理后螯合酶催化位点中的二价金属来捕获整合酶-病毒 DNA 复合物。我们最近在几篇出版物中描述了赋予临床耐药性的整合酶突变体的生化酶活性和药物敏感性。这些相同的突变体现在可用于测试我们的新型抑制剂。我们正在将这些研究扩展到整合酶柔性环中的双突变体，这种突变体通常出现在拉替拉韦耐药的患者中。工作假设是，在整合酶因第一个突变的存在而变得有缺陷后，第二个突变作为功能增益来挽救整合酶的生化活性。目的之一是了解这种互补的分子机制以及柔性环、病毒和宿主 DNA 以及抑制剂之间的结构联系。值得注意的是，我们发现柔性环双突变体140S-148H对拉替拉韦和埃替拉韦具有交叉耐药性。另一方面，143Y突变体主要对拉替拉韦具有耐药性，对埃替拉韦具有最低程度的耐药性。这些结果为因突变 143Y 而对拉替拉韦产生耐药性的患者使用埃替拉韦提供了依据（但突变 140S-148H 则不然）。在建立了一系列重组突变整合酶的检测方法后，我们能够发现新的化学型来克服拉替拉韦和埃维拉韦耐药性。