MUTATIONS EFFECTS ON INHIBITION OF HIV PROTEASE

描述：(改编自申请者摘要)：本项目的目标 是为了了解突变改变活性的分子基础 和抑制HIV-1蛋白并导致对人类的抗药性 针对蛋白水解酶抑制剂药物的免疫缺陷病毒。该计划的目标是 项目内容如下：目标1：确定催化剂活性和 14个主要突变株的抗性突变体对抑制剂的敏感性 HIV-1 PR的立场。HIV-1PR中这14个位置的突变 多肽链解释了在临床试验中观察到的耐药性 3种上市的HIV-1 PR抑制药(萨卡尼韦、爱迪那韦、利托那韦)； 突变还会导致对其他主要抑制剂的交叉耐药性 为临床使用而开发。将确定kcat/kM和KI值 对于HIV-1的每个单残基突变形式的底物面板 PR，以及所选的多残基突变体。将对数据进行分析 通过先前建立的动力学模型进行定量。目标2：实现 确定缓蚀剂之间的络合物的晶体结构 和HIV-1 PR的耐药突变株。晶体结构将是 对选定的单部位和多部位复杂抗性突变体进行测定 到选定的临床相关的抑制剂。水晶中的差异 抗性突变体、野生型和非抗性突变体之间的结构 将进行批判性分析，并与催化剂的变化进行比较 活动性是一种其他的动力学性质.目标3：对假设进行检验 HIV-1PR的催化、抑制和耐药机制。 动力学和结构数据将为测试几个 假设：(1)抗性突变体PR的催化活性决定 突变体和稳态种群的出现顺序 突变在临床治疗中的比例；(2)基因突变的主要作用 HIV-1 PR的襟翼是捕获基质的侧链和 主要特异性部位的抑制剂；(3)大多数临床上的 HIV-1 PR的选择性耐药突变恶化了与 过渡态模板(在酶分子中)到酶的同工酶 抑制剂；(4)抑制剂药物与HIV-1 PR结合最强，具有过渡性 不同于游离酶的状态构象。

DESCRIPTION: (Adapted from applicant's Abstract): The goal of this project is to understand the molecular basis by which mutations alter the activity and inhibition of the HIV-1 Protease and lead to resistance of human immunodeficiency virus against protease inhibitor drugs. The Aims of the project are as follows: Aim 1: to determine the catalytic activities and inhibitor sensitivities of resistant mutants from 14 major mutation positions of HIV-1 PR. The mutations at these 14 positions in the HIV-1 PR polypeptide chain account for the resistance observed in clinical trials of 3 marketed HIV-1 PR inhibitor drugs (Saquanivir, Idinavir, Ritonavir); the mutations also lead to cross-resistance to other major inhibitors under development for clinical use. The kcat/Km and Ki values will be determined for a panel of substrates for each single-residue mutant form of the HIV-1 PR, and for selected multiple-residue mutants. The data will be analyzed quantitatively by a previously established kinetic model. Aim 2: To determine the crystallographic structure of the complexes between inhibitors and resistance mutants of the HIV-1 PR. Crystallographic structures will be determined for chosen single and multiple-site resistant mutants complexed to selected clinically-relevant inhibitors. The differences in the crystal structures between resistant mutants, wild-type, and non-resistant mutants will be critically analyzed and compared to the change in catalytic activities a other kinetic properties. Aim 3: To test hypotheses on the mechanisms of HIV-1 PR catalysis, inhibition, and resistance to inhibitors. Kinetic and structural data will set the basis for testing several hypotheses: (1) Catalytic activities of the resistant mutant PR's determine the order of appearance of the mutants and the steady-state population ratios of the mutants during clinical therapy; (2)A major function of the flaps of the HIV-1 PR is the capturing of the sidechains of substrates and inhibitors at the major specificity positions; (3) Most of the clinically selected resistant mutations of HIV-1 PR worsen the binding of the transition state template (in the enzyme molecule) to the isostere of the inhibitors; (4) Inhibitor drugs bind strongest to HIV-1 PR with a transition state conformation that differs from that of the free enzyme.