MUTATIONS EFFECTS ON INHIBITION OF HIV PROTEASE

描述：（改编自申请人的摘要）：本项目的目标 是了解突变改变活性的分子基础 和抑制HIV-1蛋白酶，并导致人类耐药性 免疫缺陷病毒蛋白酶抑制剂药物。 的目标 目标1：确定催化活性， 14个主要突变耐药突变体对抑制剂敏感性 在HIV-1 PR的这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抑制剂药物（沙奎那韦、依地那韦、利托那韦）; 突变还导致对其他主要抑制剂的交叉耐药性， 开发用于临床。 将确定kcat/Km和Ki值 对于HIV-1的每个单残基突变形式的一组底物， PR，和选择的多残基突变体。 将对数据进行分析 通过先前建立的动力学模型定量。 目标2： 确定抑制剂之间复合物的晶体结构 和HIV-1 PR的抗性突变体。 确定所选的单位点和多位点耐药突变体， 选择临床相关抑制剂。 晶体中的差异 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， 其他动力学性质。 目标3：检验关于 HIV-1 PR催化、抑制和对抑制剂耐药的机制。 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 突变体出现的顺序和稳态种群 （2）临床治疗过程中突变体的比例;（3）突变体的主要功能 HIV-1 PR的侧翼是捕获底物的侧链， 抑制剂的主要特异性位置;（3）大多数临床 HIV-1 PR的选择性耐药突变会恶化HIV-1 PR与HIV-1受体的结合， 过渡态模板（在酶分子中）的电子等排体 抑制剂;（4）抑制剂药物与HIV-1 PR结合最强， 状态构象不同于游离酶的构象。

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.