ASSAYS FOR MEASURING THE INHIBITION OF HIV-1 ENZYMES

描述（由申请人提供）： 目前批准的药物通过干扰蛋白酶（PR）或逆转录酶（RT）的酶活性来抑制HIV-1复制。艾滋病毒迅速发展耐药性的能力，加上目前抗逆转录病毒疗法的毒性问题，需要开发其他类别的抗病毒药物。整合酶（Integrin ase，IN）作为HIV-1复制的必需酶，是一个很有吸引力的抗病毒靶点。最近的技术进步导致了良好的先导化合物的鉴定，可以作为基于结构的整合酶抑制剂（INI）设计的起点。其中一些化合物的临床评价正在进行中。该项目的总体目标是开发快速、灵敏的表型和基因型检测方法，以评估HIV-1分离株对所有HIV-1病毒酶抑制剂的敏感性，特别强调INI。我们将构建两种可供选择的载体系统，以产生含有来自患者分离株的HIV-1 pol蛋白的病毒颗粒。这些检测将基于PhenoSense HIVTM的技术，PhenoSense HIVTM是一种现有的基于细胞的抗病毒药物敏感性检测，用于评估PR和RT抑制剂。在第一阶段完成后，我们将能够为开发任何pol酶功能抑制剂的制药公司提供仅供研究使用（RUO）的检测。除了通过体外表征耐药性来帮助发现和开发第一代INI外，RUO测定还将通过针对耐药性测试载体库筛选新药候选物来帮助发现和开发对耐药菌株有活性的第二代药物。此外，RUO检测将允许在研究药物的临床评价期间监测IN抑制剂耐药性HIV-1的发展。表征这些RUO检测试剂盒在I期的性能将有助于其作为项目II期的临床验证。用于选择有效的抗逆转录病毒治疗方案，包括活性PR，RT和IN抑制剂的组合。

DESCRIPTION (provided by applicant): Currently approved drugs inhibit HIV-1 replication by interfering with the enzymatic activities of either protease (PR) or reverse transcriptase (RT). The ability of HIV to rapidly evolve drug resistance, together with the toxicity problems of current antiretroviral regimens, requires the development of additional classes of antiviral drugs. As an essential enzyme for HIV-1 replication, Integrase (IN) is an attractive antiviral target. Recent technological advances have resulted in the identification of good lead compounds that could serve as the starting point for structure-based Integrase Inhibitor (INI) design. Clinical evaluation of some of these compounds is underway. The overall goal of this project is to develop rapid, sensitive phenotypic and genotypic assays to evaluate the susceptibility of HIV-1 isolates to inhibitors of all HIV-1 viral enzymes, with particular emphasis on INIs. We will construct two alternative vector systems that produce virus particles containing HIV-1 pol proteins derived from patient isolates. These assays will be based on the technology underlying PhenoSense HIVTM, an existing cell-based antiviral drug susceptibility assay that evaluates PR and RT inhibitors. At the completion of Phase I, we will be able to provide Research Use Only (RUO) assays to pharmaceutical companies developing inhibitors of any pol enzymatic function. In addition to aiding in the discovery and development of first generation INIs by characterizing resistance in vitro, the RUO assays will assist in the discovery and development of second-generation drugs that are active against drug resistant strains by screening new drug candidates against a library of resistance test vectors. Additionally, the RUO assays will allow monitoring the development of IN inhibitor resistant HIV-1 during the clinical evaluation of investigational agents. Characterizing the performance of these RUO assays during Phase I will facilitate their clinical validation as Phase II of the project. For the selection of potent antiretroviral treatment regimens that include combinations of active PR, RT and IN inhibitors.