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。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。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 逆转录酶（RT）。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) 通过变构机制发挥活性[1]。最近抑制RNA的药物 RT 的去除函数（RNH）不影响聚合酶活性也已被 发现了。不幸的是，由于所有这些药物的耐药性迅速发展 与HIV病毒的高突变率有关。残留变化可能会消除有利的 结合相互作用，或者它们可能通过空间效应阻止药物进入。他们可能 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。可以预测和量化局部的数学模型 突变对药物结合和催化活性的远程影响可能导致 对抗耐药性的新策略。 HIV-1 RT 结合的初步研究 抑制剂二羟基苯甲酰萘腙（DHBNH）表明这是可行的。这 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)。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 药物结合和催化位点的突变。之前尝试使用 QM 来实现此目的 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]。 QMROI 方法旨在创建一个更 具有完整多肽链的真实局部结合环境。这样一个 环境有更好的机会识别导致的构象变化 到耐药性。 QMROI 以结合位点为中心，包括 结合的药物和所有含有位点中心 9 ¿ 范围内的原子的残基。 根据需要添加残基以创建一组短的连续多肽 定义结合位点的链。这些链的末端被氢封端 使开放价饱和。这是通过在 N 末端突变来实现的 氨基氮转化为氢。 C 末端的羰基碳发生突变 到氢气。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 周围的蛋白质。 QMROI 中的所有剩余原子均不受约束。这 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。就 RT 而言， QMROI 包含约 400-500 个原子。这个 QMROI 足够大，可以包含所有 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 足以影响药物结合位点。它也足够大，可以捕获 高度保守的酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸 (YMDD) 基序 聚合酶催化位点。每个 QMROI 结构的几何形状由下式确定 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]。所有计算均使用 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。结合能计算为 结合药物的蛋白质的总能量和蛋白质的总能量和 自己下药。进行频率计算以获得零点 能量修正和热力学函数。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。 QMROI 原子可用的构象状态是 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。测量 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”。这样的 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 模式。 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 溶剂环境。 QMROI 模型提供了有关 突变引起的药物结合位点的空间变化。在很多情况下，这 无法通过纯粹的实验方法获得信息。详细的 有关药物结合位点几何关系的信息对于 合理的药物设计。尽管QMROI模型的计算强度很大 从角度来看，这种方法适合使用并行处理的大规模生产 现代集群。项目初期的实验设计重点 在两个感兴趣的区域上。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)。这两个结合位点均邻近 RT 聚合酶催化位点 并且两个结合位点都有重叠的成分。在这两种情况下也有绑定 涉及“诱导契合”。更重要的是，QMROI 区域都与 聚合酶催化位点中的关键 YMDD 基序。每个 QMROI 的几何形状将 最初是在没有突变的情况下进行优化的。由定义的两种构象状态 将研究这两种药物的 QMROI 中固定帽原子的位置。这些 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）。将比较两个状态之间的 YMDD 主题。 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 组中不存在或在 RNH 组中最少。基线 QMROI 区域也将 在不存在抑制剂药物的情况下研究每个结合位点。这将 可以使用 2i5j 的 25 ns MD 模拟中可用的构象来完成 在不含 DHBNH 的清水中。该模拟是作为试点的一部分进行的 研究探索这种方法的可行性。分析结合时 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。这 突变将从已知赋予的突变列表中选择 奈韦拉平耐药。赋予 DHBNH 抗性的突变尚未被证实 确定。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 为野生型计算相应的参数。几何变化 还将分析结合和未结合状态的 YMDD 基序，以确定 药物结合对聚合酶催化位点的影响。分析将提供 深入了解每个突变所赋予的耐药机制。更多的 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。第二阶段 该项目将把计算机 QMROI 研究与实验方法结合起来。这将 通过与 Michael Parniak 实验室的合作来完成 匹兹堡大学。该实验室专注于新型RT的开发 靶向 RNH 位点的抑制剂 [4]。 QMROI 方法将用于研究 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。此类研究还将用于指导定点诱变研究 HIV-1 耐药性。 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. 何霞，梅艳，向艳， 张大文、张建中：HIV-1耐药性的量子计算分析 通过点突变将酶逆转录为奈韦拉平。蛋白质：结构， 功能与生物信息学，61：423-432，2005。3. Kohn W，Sham LJ：量子 非均匀电子气中的密度振荡。物理。修订版，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 腙束缚在一个新地点。美国化学学会化学生物学，1：702-711，2006。