PROTEASE INHIBITOR DESIGN AGAINST MDR HIV VARIANTS

描述（由申请人提供）： 对现有蛋白酶抑制剂具有耐药性的HIV病毒株的发展是成功的长期抗逆转录病毒治疗的主要障碍之一。我们正在研究HIV-1蛋白酶的结构变化，这些蛋白酶来自对许可的蛋白酶抑制剂具有抗性的自然发生的临床变体。该项目的长期目标是研究艾滋病毒蛋白酶耐药性的结构基础，并利用这些知识制定克服耐药性的战略。正在测试的假设是，HIV-1蛋白酶多药耐药性与扩大蛋白酶活性位点空腔的突变有关，因此抑制剂以较低的亲和力与酶的变体形式结合。 该项目的短期目标是对一组四种多药耐药HIV-1蛋白酶临床分离株进行结构-功能研究。为此，我们将： 目标1。定义MDR HIV-1蛋白酶变体中多药耐药性的结构基础，并对MDR HIV-1蛋白酶变体进行功能分析，以开发三维结构-活性关系，从蛋白质-配体相互作用方面解释为什么药物抑制这些结构不如野生型。 目标2.设计、合成和测试针对MDR HIV-1蛋白酶变体的小分子抑制剂。 将对目标1中描述的HIV-1蛋白酶变体进行X射线晶体学和功能研究。结构研究的重点是发现蛋白酶变体的三维变化。将突变蛋白酶-配体结构与野生型蛋白酶-配体结构进行比较。将使用表面等离子体共振（BIACORE）、等温滴定量热法（ITC）、酶测定和病毒学药物敏感性测定对改变的蛋白酶变体的配体进行功能研究。代表抗MDR HIV-1蛋白酶的先导化合物的小分子配体将被鉴定、合成并测试功效。肽衍生物和拟肽配体将被构建成四类：修饰肽、反转肽、类肽和酰胺电子等排体。解决为什么某些突变的HIV-1蛋白酶不被标准抑制剂抑制的结构问题，并利用这些信息来设计新的有效化合物，对人类福利非常重要。

DESCRIPTION (provided by applicant): The development of HIV virus strains that are resistant to existing protease inhibitors is one of the major obstacles to successful long-term antiretroviral therapy. We are investigating the structural changes of HIV-1 proteases from naturally occurring clinical variants that have become resistant to licensed protease inhibitors. The long-term goal of this project is to study the structural basis of HIV protease drug resistance and to use that knowledge to devise strategies to overcome resistance. The hypothesis being tested is that HIV-1 protease multidrug resistance is associated with mutations that expand the active site cavity of the protease, and as a result the inhibitors bind with lower affinity to the variant forms of the enzyme. The short-term goal of the project is to perform structure-function studies with a set of four multidrug-resistant HIV-1 protease clinical isolates. To accomplish this we will: Aim 1. Define the structural basis for multi-drug resistance in MDR HIV-1 protease variants and perform functional analyses of MDR HIV-1 protease variants to develop three dimensional structure-activity relationships that explain, in terms of protein-ligand interactions, why the drugs inhibit these structures less well compared to the wild-type. Aim 2. Design, synthesize and test small molecule inhibitors against the MDR HIV-1 protease variants. The HIV-1 protease variants described in Aim 1 will be subjected to X-ray crystallographic and functional studies. Structural studies are focused at discovering the three-dimensional changes in the protease variants. Mutant protease-ligand structures will be compared to wild-type protease-ligand structures. Functional studies of the ligands to the altered protease variants will be performed using surface plasmon resonance (BIACORE), isothermal titration calorimetry (ITC), enzyme assays and virologic drug susceptibility assays. Small molecule ligands that represent lead compounds against the MDR HIV-1 protease will be identified, synthesized and tested for efficacy. Peptide derivatives and peptidomimetic ligands will be constructed in four categories: modified peptides, retro-inverso peptides, peptoids and amide isosteres. Solving the structural problem of why certain mutant HIV-1 proteases are not inhibited by the standard inhibitors and using this information to design new and effective compounds are of a great importance to human welfare.