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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。主题项目和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 大多数针对HIV感染干扰病毒有效的药物 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） 通过变构机制的活动[1]。最近抑制RNA的药物 RT的去除功能（RNH）也没有影响聚合酶活性 发现。不幸的是，耐药性迅速发展为所有这些代理 达到HIV病毒的高突变率。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 还要干扰柔韧性，以防止在结合位点“诱发拟合”，否则它们可能会 改变变构效应。可以预测和量化本地的数学模型 突变对药物结合和催化活性的远程影响可能导致 打击耐药性的新策略。有关HIV-1 RT的试点研究 抑制剂二羟基苯甲酰二酰基羟基羟基（DHBNH）表明这是可行的。这 基本方法涉及将量子机械（QM）计算应用到 分析选定的感兴趣区域（QMROI）。主要想法是创建一个“量子” 机械实验室”可以在硅中扰动，以建模 药物结合和催化位点的突变。以前尝试使用QM 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方法试图创建更多 现实的局部结合环境，具有完整的多肽链。这样的 环境有更好的机会确定构象变化的领先 具有耐药性。 QMROI以绑定位点为中心，包括 在该地点中心的9`绑定药物和所有包含原子的残留物。 根据需要添加残留物以创建一组短的连续多肽 定义结合位点的链。这些链的末端用氢盖了 饱和开放价。这是通过突变在N末端完成的 氨基氮氮至氢。在C末端，羰基碳被突变 到氢。这些氢“盖”原子的位置在几何过程中是固定的 优化以锁定对QMROI施加的构象状态 周围蛋白质。 QMROI中的所有剩余原子都是不受限制的。这 通过优化一组，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。在RT的情况下 QMROI包含约400-500个原子。这个Qmroi足够大，可以包括所有 结合药物中的原子和所有残留物具有近距离原子的原子 足以影响药物结合位点。它也足够大，可以捕获 高度配置的酪氨酸 - 甲硫代硫代天冬氨酸天冬氨酸（YMDD）主题 聚合酶催化位点。每个QMROI结构的几何形状由 其分子波函数在密度函数理论（DFT）上的数值解 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。绑定能量是通过应用确定的 用于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 蛋白质的总能量与结合药物以及蛋白质的总能量 独自吸毒。进行频率计算以获得零点 能量校正和热力学功能。突变对药物的影响 通过更换硅中的住所侧chain，然后是新的，然后是新的，然后是新的 QMROI计算。 QMROI原子可用的构象状态是 通过改变固定的固定氢帽原子的位置来模拟 定义QMROI的多肽链的末端。允许方差 在这些固定盖原子之间的成对位置，由 位置变异在不同的晶体学结构中观察到的分子 动力学（MD）模拟或粗粒模型，例如各向异性弹性 网络模型（ANM）。 QMROI模型提供了确定效果的手段 使用电子结构计算对药物结合的任何突变。测量 在催化结合位点中关键氨基酸基序中产生的失真，提供了 给定突变体的“适应性”来执行其催化功能。这样的 可以根据原子位移来量化扭曲 胡椒骨架原子的二面角或氢键变化 模式。 QMROI模型代表了第一种量子机械方法 HIV耐药性问题，该问题解决了局部和全球药物结合能的问题 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 由突变诱导的药物结合位点的空间改变。在许多情况下，这个 信息无法通过纯粹的实验方法获得。详细的 关于药物结合位点几何关系的信息对于 理性药物设计。即使QMROI模型从计算中很强烈 角度，这种方法适用于使用并行处理中的大规模生产 现代集群。项目初始阶段的实验设计集中在 在两个感兴趣的地区。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 并且两个绑定位点都有重叠的组件。在这两种情况下也具有约束力 涉及“诱导的拟合”。更重要的是，QMROI地区都重叠 聚合酶催化位点中的临界YMDD基序。每个Qmroi的几何形状将 最初在没有突变的情况下进行优化。两个由 QMROI中固定帽原子的位置将用于两种药物的研究。这些 状态将表示固定之间的最大和最小成对分离 根据对可用晶体学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图案。 如果该基序的失真是NNRTI抑制的基础，则应最大 RNH集合中的NNRTI集合和不存在或最小。基线QMROI地区也将 在不存在抑制剂药物的情况下对每个结合位点进行研究。这会 可以使用25 ns MD模拟2i5j的构象来完成 在没有DHBNH的显式水中。该模拟是作为飞行员的一部分进行的 研究探讨了这种方法的可行性。当分析结合 对于两种药物和两个基线区域，能量和YMDD失真都是完整的， 完整的组将通过七个不同的点突变恢复。 将从会议已知的突变列表中选择突变 奈韦拉平的抗性。突变会议DHBNH的抵抗还没有 确定。所考虑的突变将是L100I，K103N，V106A，V108I，Y181C， Y188H和G190S [1]。结合能，几何改变和变化 将为每个突变体计算的临界YMDD基序将与 针对野生类型计算的相应参数。几何改变 还将分析边界和未结合状态中的YMDD图案以确定 药物结合对聚合酶催化位点的影响。分析将提供 洞悉每个突变赋予的抗性机制。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 可以用来用于制定类似物的合理设计。第二阶段 项目将与实验方法结合使用硅QMROI研究。这会 可以通过与迈克尔·帕尼亚克（Michael Parniak）的实验室合作完成 匹兹堡大学。该实验室的重点是开发新的RT 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，以指导这种设计 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位的诱变研究 HIV-1耐药性。 1。IlinaT，帕尼亚克MA：HIV-1的抑制剂反向 转录酶。药理学的进步，56：121-167，2008。2。他X，Mei Y，Xiang Y， 张DW，张JZ：HIV-1耐药性的量子计算分析 通过点突变逆转录酶到奈韦拉平。蛋白质：结构， 功能与生物信息学，61：423-432，2005。3。科恩W，假LJ：量子 不均匀电子气体中的密度振荡。物理。修订版，137（6a）：1697- 1705，1965。4。Himmel DM，Sarafinos SG，Dharmasina S，Parniak MA等：HIV-1 带有RNase H抑制剂二羟基苯甲酰甲酰基的逆转录酶结构 挂索在一个新颖的地点绑定。 ACS化学生物学，1：702-711，2006。