A QUANTUM MECHANICAL APPROACH FOR EXPLORING HIV DRUG RESISTANCE

探索 HIV 耐药性的量子力学方法

• 批准号: 8171876
• 负责人: JOHN Kenric VRIES
• 金额: $ 0.14万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171876
• 关键词: Address; Affect; Amino Acid Motifs; Amino Acids; Aspartate; Binding; Binding Sites; Bioinformatics; Biology; Carbon; Catalytic Domain; Cereals; Charge; Chemicals; Collaborations; Computer Analysis; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Databases; Development; Drug Binding Site; Drug Design; Drug resistance; Electrons; Electrostatics; Environment; Excision; Experimental Designs; Frequencies; Funding; Gases; Grant; HIV; HIV Infections; HIV drug resistance; HIV-1 Reverse Transcriptase; HIV-1 drug resistance; Hydrazones; Hydrogen; Hydrogen Bonding; Induced Mutation; Institution; Laboratories; Lead; Measurement; Measures; Mechanics; Methionine; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; Nevirapine; Nitrogen; Nucleosides; Pattern; Peptides; Pharmaceutical Preparations; Pharmacology; Phase; Pilot Projects; Point Mutation; Polymerase; Positioning Attribute; Production; Proteins; RNA; RNA-Directed DNA Polymerase; Research; Research Personnel; Resistance; Resources; Reverse Transcriptase Inhibitors; Ribonuclease H; Simulate; Site; Site-Directed Mutagenesis; Solutions; Solvents; Source; Structure; Sum; Surface; Surveys; Thermodynamics; Tyrosine; United States National Institutes of Health; Universities; Variant; Vertebral column; Viral; Water; analog; base; combat; density; design; electronic structure; fitness; flexibility; inhibitor/antagonist; insight; interest; mathematical model; molecular dynamics; mutant; network models; nevirapine resistance; non-nucleoside reverse transcriptase inhibitors; novel; parallel processing; polypeptide; prevent; programs; protein structure function; quantum; resistance mechanism; simulation; theories

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The majority of drugs that are effective against HIV infection interfere with viral reverse transcriptase (RT). These drugs include nucleoside reverse transcriptase inhibitors (NRTI) that directly interfere with the polymerase catalytic site in RT and non-nucleoside reverse transcriptase inhibitors (NNRTI) that influence polymerase activity through an allosteric mechanism [1]. Recently drugs that inhibit the RNA removal function (RNH) of RT without affecting polymerase activity have also been discovered. Unfortunately, drug resistance develops rapidly to all these agents due to the high mutation rate of the HIV virus. Residue changes may eliminate favorable binding interactions or they may block drug access through steric effects. They may also interfere with flexibility preventing "induced fits" at the binding site or they may alter allosteric effects. A mathematical model that could predict and quantify the local and remote effects of mutations on drug binding and catalytic activity could lead to new strategies for combating drug resistance. Pilot studies on HIV-1 RT bound to the inhibitor dihydroxy benzoyl napthyl hydrazone (DHBNH) indicate this is feasible. The basic approach involves the application of quantum mechanical (QM) calculations to analyze selected regions of interest (QMROI). The main idea is to create a "quantum mechanical laboratory" that can be perturbed in silico to model the effects of mutations on drug binding and catalytic sites. Previous attempt to use QM for this purpose have treated drug binding as the sum of the interactions between drugs and isolated amino acid residues [2]. The QMROI approach seeks to create a more realistic local binding environment with complete polypeptide chains. Such an environment has a better chance for identifying the conformational changes leading to drug resistance. The QMROI is centered on the binding site and includes the bound drug and all residues containing atoms within 9 ¿ of the center of the site. Residues are added as necessary to create a set of short continuous polypeptide chains defining the binding site. The ends of these chains are capped with hydrogens to saturate the open valences. This is accomplished on the N-terminus by mutating the amino nitrogen to hydrogen. On the C-terminus, the carbonyl carbon is mutated to hydrogen. The positions of these hydrogen "cap" atoms are fixed during geometric optimization to lock in the conformational state imposed on the QMROI by the surrounding protein. All remaining atoms in the QMROI are unconstrained. The electrostatic effect of the surrounding protein is simulated by optimizing at set of point charges distributed on a surface surrounding the QMROI. In the case of RT, the QMROI contains ~400-500 atoms. This QMROI is large enough to include all the atoms in the bound drug and all the residues with polarizable atoms that are close enough to influence the drug binding site. It is also large enough to capture the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif in the polymerase catalytic site. The geometry of each QMROI structure is determined by numerical solution of its molecular wavefunction at a density function theory (DFT) level (b3lyp/6-31g(d,p)) of QM theory [3]. All calculations are carried out using the Gaussian'03" suite of programs. Binding energies are determined by applying frequency and single point energy studies to the drug and protein components of the optimized QMROI. The binding energy is calculated as the difference between the total energy of the protein with bound drug and the total energies of the protein and drug by themselves. Frequency calculations are carried out to obtain zero point energy corrections and thermodynamic functions. The effects of mutations on drug binding are studied by replacing the residue sidechains in silico followed by new QMROI calculations. The conformational states available to the QMROI atoms are simulated by varying the positions of the fixed hydrogen cap atoms that anchor the ends of the set of polypeptide chains that define the QMROI. The allowable variance in the pairwise positions between these fixed cap atoms is determined by the positional variation observed in different crystallographic structures, molecular dynamics (MD) simulations or coarse grained models such as the anisotropic elastic network model (ANM). The QMROI model provides a means for determining the effect of any mutation on drug binding using electronic structure calculations. Measurement of the distortion created in key amino acid motifs in catalytic binding sites provides a measure of the "fitness" of a given mutant to carry out its catalytic function. Such distortions can be quantified in terms of atomic displacements, changes in the dihedral angles of peptide backbone atoms or alterations in hydrogen bonding patterns. The QMROI model represents the first quantum mechanical approach to the problem of HIV drug resistance that addresses drug binding energy, local and global conformational change and the electrostatic effect of the surrounding protein and solvent environment. The QMROI model provides quantitative information about the steric alterations in drug binding sites induced by mutations. In many cases, this information is not available through purely experimental approaches. Detailed information about geometric relationships in drug binding sites is essential for rational drug design. Even though the QMROI model is intense from the calculation standpoint, this approach is suitable for mass production using parallel processing in modern clusters. The experimental design for the initial phase of the project focuses on two regions of interest. The first is the binding site for the NNRTI inhibitor nevirapine (PDB 1vrt). The second is the binding site for the RNH inhibitor DHBNH (2i5j). Both of these binding sites are adjacent to the RT polymerase catalytic site and both binding sites have overlapping components. Binding in both instances also involves an "induced fit". More importantly, the QMROI regions both overlap the critical YMDD motif in the polymerase catalytic site. The geometry of each QMROI will initially be optimized with no mutations. Two conformational states defined by the position of the fixed cap atoms in the QMROI will be studied for both drugs. These states will represent the maximum and minimum pairwise separation between fixed atoms estimated from a survey of the available crystallographic structures in the protein data bank (PDB). The YMDD motif between the two states will be compared. If distortion of this motif is the basis for NNRTI inhibition, it should be at a maximum in the NNRTI set and absent or minimal in the RNH set. Baseline QMROI regions will also be studied for each binding site without the presence of the inhibitor drugs. This will be accomplished using the conformations available from a 25 ns MD simulation of 2i5j in explicit water without DHBNH. This simulation was carried out as part of the pilot studies exploring the feasibility of this approach. When analysis of the binding energies and YMDD distortion is complete for both drugs and both baseline regions, the complete set will be restudied with seven different point mutations. The mutations will be selected from the list of mutations that are known to confer nevirapine resistance. Mutations conferring DHBNH resistance have not yet been identified. The mutations considered will be L100I, K103N, V106A, V108I, Y181C, Y188H and G190S [1]. Binding energies, geometric alterations and changes in the critical YMDD motif calculated for each mutant will be compared with the corresponding parameters calculated for the wild type. The geometric alterations in the YMDD motif in the bound and unbound states will also be analyzed to determine the influence of drug binding on the polymerase catalytic site. Analysis will provide insight into the mechanism of resistance conferred by each mutation. More importantly, it will provide geometric information about the drug and the binding site that can be used for the rational design of drug analogs. The second phase of the project will combine in silico QMROI studies with experimental approaches. This will be accomplished through collaborations with the laboratory of Michael Parniak at the University of Pittsburgh. This laboratory is focused on the development of new RT inhibitors that target the RNH site [4]. The QMROI approach will be used to study the effects of potential new inhibitory compounds, to guide the design of such compounds and to judge the potential effects of mutations that have not yet been observed. Such studies will also be used to guide site-directed mutagenesis studies of HIV-1 drug resistance. 1. Ilina T, Parniak MA: Inhibitors of HIV-1 Reverse Transcriptase. Advances in Pharmacology, 56:121-167, 2008. 2. He X, Mei Y, Xiang Y, Zhang DW, Zhang JZ: Quantum Computational Analysis for Drug Resistance of HIV-1 Reverse Transcriptase to Nevirapine through Point Mutations. Proteins: Structure, Function and Bioinformatics, 61:423-432, 2005. 3. Kohn W, Sham LJ: Quantum Density Oscillations in an Inhomogeneous Electron Gas. Phys. Rev., 137(6A):1697- 1705, 1965. 4. Himmel DM, Sarafinos SG, Dharmasina S, Parniak MA, et al: HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor Dihydroxy Benzoyl Naphthyl Hydrazone Bound at a Novel Site. ACS Chemical Biology, 1:702-711, 2006.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 大多数对艾滋病毒感染有效的药物都会干扰病毒 这些药物包括核苷逆转录酶。 直接干扰 RT 中聚合酶催化位点的抑制剂 (NRTI) 影响聚合酶的非核苷逆转录酶抑制剂 (NNRTI) 最近的药物通过变构机制抑制 RNA 的活性。 RT 的去除函数（RNH）不影响聚合酶活性也已被 不幸的是，由于所有这些药物的耐药性迅速发展。 对HIV病毒的高突变率可能有利的消除残留变化。 结合相互作用，或者它们可能通过空间效应阻止药物进入。 也会干扰灵活性，防止结合位点处的“诱导配合”，否则它们可能 改变变构效应的数学模型可以预测和量化局部效应。 突变对药物结合和催化活性的远程影响可能导致 HIV-1 RT 与抗药性作斗争的新策略。 抑制剂二羟基苯甲酰萘腙（DHBNH）表明这是可能的。 基本方法涉及应用量子力学（QM）计算 分析选定的感兴趣区域（QMROI）的主要思想是创建一个“量子”。 机械实验室”可以在计算机中受到扰动以模拟 之前尝试使用 QM 来实现这一点。 目的将药物结合视为药物之间相互作用的总和 和分离的氨基酸残基 [2]。 具有完整多肽链的真实局部结合环境。 环境有更好的机会识别导致的构象变化 QMROI 以结合位点为中心，包括 结合药物和所有含有 9 ¿ 以内原子的残基网站的中心。 根据需要添加残基以产生一组短的连续多肽 定义结合位点，这些链的末端被氢封端。 使开放价态饱和，这是通过在 N 末端突变来实现的。 在C末端，氨基氮变为氢。 这些氢“帽”原子的位置在几何过程中是固定的。 优化以锁定 QMROI 上的构象状态 QMROI 中的所有剩余原子均不受约束。 通过优化一组来模拟周围蛋白质的静电效应 点电荷分布在 QMROI 周围的表面上。 QMROI 包含约 400-500 个原子。这个 QMROI 足够大，可以包含所有原子。 结合药物中的原子以及所有具有接近的可极化原子的残基 它也足够大以捕获药物结合位点。 高度保守的酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸 (YMDD) 基序 每个 QMROI 结构的几何形状由下式决定。 其分子波函数在密度函数理论 (DFT) 下的数值解 QM 理论的水平 (b3lyp/6-31g(d,p)) [3]。 Gaussian'03" 程序套件。结合能通过应用来确定 对药物和蛋白质成分的频率和单点能量研究 优化的 QMROI 计算为结合能之间的差异。 结合药物的蛋白质的总能量和蛋白质的总能量和 药物本身进行频率计算以获得零点。 能量校正和热力学函数的突变对药物的影响。 通过在计算机中替换残基侧链，然后用新的侧链来研究结合 QMROI 计算可用于 QMROI 原子的构象状态为 通过改变固定氢帽原子的位置来模拟 定义 QMROI 的一组多肽链的末端 允许的方差。 这些固定帽原子之间的成对位置由 在不同晶体结构、分子中观察到的位置变化 动力学 (MD) 模拟或粗粒度模型，例如各向异性弹性 网络模型 (ANM)。QMROI 模型提供了一种确定效果的方法。 使用电子结构计算来确定药物结合的任何突变。 催化结合位点中关键氨基酸基序产生的扭曲提供了 衡量给定突变体执行其催化功能的“适应性”。 扭曲可以用原子位移、原子的变化来量化 肽主链原子的二面角或氢键的改变 QMROI 模型代表了第一个量子力学方法。 解决局部和全球药物结合能的艾滋病毒耐药性问题 周围蛋白质的构象变化和静电效应 QMROI 模型提供有关溶剂环境的定量信息。 在许多情况下，由突变引起的药物结合位点的空间变化。 信息无法通过纯粹的实验方法获得。 有关药物结合位点几何关系的信息对于 尽管 QMROI 模型的计算结果很强烈。 立场，这种方法适合使用并行处理的大规模生产 项目初期阶​​段的实验设计重点是现代集群。 第一个是 NNRTI 抑制剂的结合位点。 奈韦拉平 (PDB 1vrt) 第二个是 RNH 抑制剂 DHBNH 的结合位点。 (2i5j) 这两个结合位点都与 RT 聚合酶催化位点相邻。 并且两个绑定站点都具有重叠的实例组件。 更重要的是，QMROI 区域都与“诱导拟合”重叠。 聚合酶催化位点中的关键 YMDD 基序将影响每个 QMROI 的几何形状。 最初是在没有突变的情况下进行优化的。 将研究这两种药物的 QMROI 中固定帽原子的位置。 状态将表示固定之间的最大和最小成对间隔 通过对现有晶体结构的调查估计的原子 蛋白质数据库（PDB）将比较两种状态之间的 YMDD 基序。 如果这个基序的扭曲是 NNRTI 抑制的基础，那么它应该在最大 NNRTI 组和 RNH 组中的基线 QMROI 区域不存在或最少。 在不存在抑制剂药物的情况下研究每个结合位点。 可以使用 2i5j 的 25 ns MD 模拟中可用的构象来完成 该模拟是作为试点的一部分进行的。 研究探讨了这种方法在分析结合时的可行性。 两种药物和两个基线区域的能量和 YMDD 失真都是完整的， 整个集合将用七个不同的点突变进行重新研究。 突变将从已知的突变列表中选择 尚未发现奈韦拉平耐药性突变。 所考虑的突变为 L100I、K103N、V106A、V108I、Y181C、 Y188H 和 G190S [1] 结合能、几何变化和变化。 为每个突变体计算的关键 YMDD 基序将与 野生型的相应参数。 还将分析结合和未结合状态的 YMDD 基序，以确定 分析药物结合对聚合酶催化位点的影响。 深入了解每种突变所赋予的耐药机制。 重要的是，它将提供有关药物和结合位点的几何信息 可用于药物类似物的合理设计。 该项目将把 QMROI 研究与实验方法结合起来。 通过与 Michael Parniak 实验室的合作来完成 匹兹堡大学该实验室专注于新 RT 的开发。 QMROI 方法将用于研究 RNH 位点的抑制剂。 潜在的新抑制化合物的影响，以指导此类的设计 化合物并判断尚未发生的突变的潜在影响 观察到的此类研究还将用于指导定点诱变研究。 HIV-1 耐药性的研究 1. Ilina T, Parniak MA：HIV-1 逆转抑制剂。 转录酶。药理学进展，56：121-167，2008。2.何X，梅Y，向Y， 张大文、张建中：HIV-1耐药性的量子计算分析 通过蛋白质点突变逆转录为奈韦拉平：结构， 功能和生物信息学，61：423-432，2005。3. Kohn W，Sham LJ：量子 非均匀电子气体中的密度振荡。物理评论，137(6A)：1697- 1705, 1965. 4. Himmel DM, Sarafinos SG, Dharmasina S, Parniak MA 等人：HIV-1 具有 RNase H 抑制剂二羟基苯甲酰萘的逆转录酶结构 腙结合在新位点。ACS 化学生物学，1：702-711，2006 年。