STRUCTURE-FUNCTION STUDY OF HIV PROTEASE DRUG RESISTANCE

该项目的长期目标是调查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策略。正在测试的新假设是，HIV-1蛋白酶耐药性是由蛋白酶活性位点扩大的突变引起的，因此抑制剂与该酶的变体形式结合的亲和力较低。本项目的短期目标是采用结构、病毒学和生化相结合的方法研究4个多重耐药HIV蛋白酶临床分离株的结构和功能。为实现这一目标，我们将：利用x射线晶体学和分子模拟技术分析了四种多重耐药“特征突变体”（807,3761,1385,769）抑制剂复合物的结构。将系统地比较30种蛋白酶变异体配体复合物的模型结构，以确定抑制剂或底物结合的变化。三分之一的模型结构将通过x射线晶体学进行验证。目标2。测定一系列四种多重耐药HIV- 1蛋白酶（807、3761、1385、769）的病毒学（IC50）和酶学（Ki、Km、Vmax）耐药指标，并将功能结果与Aim 1获得的结构信息相关联。来自四个多重耐药临床分离株的结构-功能数据将用于检验耐药假设，包括新的HIV蛋白酶活性位点扩展假设。如果活性位点扩展假说得到证实，它将为克服耐药性提供一种合乎逻辑的方法。解决为什么一些突变的HIV-1蛋白酶不能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的化合物，对人类的福祉具有重要意义。

The long-term goal of this project is to investigate the structural basis of HIV protease drug resistance and to use that knowledge to devise strategies to overcome resistance. The novel hypothesis being tested is that HIV-1 protease drug resistance is caused by mutations that expand the active site 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 this project is to study the structure and function of four multiple drug resistant HIV protease clinical isolates by using a combined structural, virological and biochemical approach. To accomplish this we will: Aim 1. Analyze the structures of inhibitor complexes of four multiple drug resistant "signature mutants" (807, 3761, 1385, 769) by using X-ray crystallographic and molecular modeling techniques. 30 modeled structures of protease variant-ligand complexes will be systematically compared to determine changes in inhibitor or substrate binding. One-third of the modeled structures will be verified by X-ray crystallography. Aim 2. Measure the virological (IC50) and enzymatic (Ki, Km, Vmax) indicators of drug resistance for a series of four multiple drug resistant HIV- 1 proteases (807, 3761, 1385, 769) and correlate the functional results with structural information derived from Aim 1. The structure-function data from the four multiple drug resistant clinical isolates will be used to examine drug resistance hypotheses including the novel HIV protease active site expansion hypothesis. If the active site expansion hypothesis is confirmed, it will suggest a logical approach to overcome drug resistance. Solving the structural problem of why some mutant HIV-1 proteases cannot be inhibited by the standard inhibitors and using this information to design new and effective compounds is of great importance to human welfare.