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: 7956337
• 负责人: JOHN Kenric VRIES
• 金额: $ 0.08万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956337
• 关键词: Address; Affect; Amino Acid Motifs; Amino Acids; Aspartate; Binding; Binding Sites; Bioinformatics; Biology; Biomedical Research; 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; High Performance Computing; 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。 大多数有效对抗 HIV 感染的药物都会干扰病毒逆转录酶 (RT)，这些药物包括直接干扰 RT 聚合酶催化位点的核苷逆转录酶抑制剂 (NRTI) 和直接干扰 RT 聚合酶催化位点的非核苷逆转录酶抑制剂 (NNRTI)。通过变构机制影响聚合酶活性[1]，最近还发现了抑制 RT 的 RNA 去除功能（RNH）而不影响聚合酶活性的药物，不幸的是，所有这些药物的耐药性迅速发展。由于 HIV 病毒的高突变率，残基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，它们也可能干扰灵活性，防止结合位点的“诱导配合”，或者它们可能改变变构效应。可以预测和量化突变对药物结合和催化活性的局部和远程影响的数学模型可能会导致针对与抑制剂二羟基苯甲酰基萘腙结合的 HIV-1 RT 进行抗耐药性的新策略。 （DHBNH）表明这是可行的，其基本方法涉及应用量子力学（QM）计算来分析选定的感兴趣区域（QMROI），其主要思想是创建一个可以在计算机中进行扰动的“量子力学实验室”。先前尝试使用 QM 来模拟突变对药物结合和催化位点的影响，将药物结合视为药物与分离的氨基酸残基之间相互作用的总和 [2]。具有完整多肽链的更真实的局部结合环境，这样的环境有更好的机会识别导致耐药性的构象变化。QMROI 以结合位点为中心，包括结合的药物和 9 ¿根据需要添加残基以创建一组定义结合位点的短连续多肽链，这些链的末端用氢封端以使开放价饱和。在 C 末端，羰基碳突变为氢。这些氢“帽”原子的位置在几何优化过程中被固定，以锁定 QMROI 上的构象状态。 QMROI 中的所有剩余原子均不受约束 通过优化分布在 QMROI 周围表面上的点电荷集来模拟周围蛋白质的静电效应。该 QMROI 足够大，可以包含结合药物中的所有原子以及足够接近以影响药物结合位点的所有具有可极化原子的残基。聚合酶催化位点中保守的酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸 (YMDD) 基序每个 QMROI 结构的几何形状由其分子波函数在密度函数理论 (DFT) 水平 (b3lyp/6-31g(d) 的数值解确定。 QM 理论 [3] 的所有计算均使用 Gaussian'03" 程序套件进行。结合能通过应用频率和单次确定。对优化的 QMROI 的药物和蛋白质成分进行点能量研究 结合能计算为结合药物的蛋白质的总能量与蛋白质和药物本身的总能量之间的差值。通过在计算机中替换残基侧链，然后通过改变 QMROI 原子的位置来模拟可用的构象状态，从而获得零点能量校正和热力学函数。锚定定义 QMROI 的一组多肽链末端的固定氢帽原子 这些固定帽原子之间的成对位置的允许差异由在不同晶体结构、分子动力学 (MD) 模拟或中观察到的位置变化决定。粗粒度模型，例如各向异性弹性网络模型 (ANM)，提供了一种使用电子结构计算来确定任何突变对药物结合的影响的方法。催化结合位点提供了对给定突变体执行其催化功能的“适应性”的测量，这种扭曲可以通过原子位移、肽主链原子二面角的变化或氢键模式的改变来量化。该模型代表了解决 HIV 耐药性问题的第一个量子力学方法，该方法解决了药物结合能、局部和整体构象变化以及周围蛋白质和溶剂环境的静电效应。模型提供了有关突变引起的药物结合位点空间变化的定量信息，尽管 QMROI 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详细信息。从计算的角度来看，该模型是密集的，该方法适合在现代集群中使用并行处理进行大规模生产。该项目初始阶段的实验设计重点关注两个感兴趣的区域，第一个是 NNRTI 抑制剂奈韦拉平的结合位点。 （PDB第二个是 RNH 抑制剂 DHBNH (2i5j) 的结合位点，这两个结合位点都与 RT 聚合酶催化位点相邻，并且两个结合位点都有重叠的成分。更重要的是，QMROI 区域都与聚合酶催化位点中的关键 YMDD 基序重叠。每个 QMROI 的几何形状最初将被优化，没有由 位置定义的两种构象状态。将研究两种药物的 QMROI 中的固定帽原子，这些状态将代表根据蛋白质数据库 (PDB) 中可用晶体结构的调查估计的固定原子之间的最大和最小成对间隔。如果该基序的变形是 NNRTI 抑制的基础，则在 NNRTI 组中该基序的变形应为最大，并且在 RNH 组中不存在或最小，还将研究每个结合位点的基线 QMROI 区域。这将使用 2i5j 在不含 DHBNH 的显性水中进行的 25 ns MD 模拟获得的构象来完成，该模拟是探索该方法可行性的试点研究的一部分。如果两种药物和两个基线区域的结合能和 YMDD 畸变都已完成，则将从已知赋予奈韦拉平耐药性的突变列表中选择七个不同的点突变来重新研究完整的集合。 DHBNH 抗性尚未确定，将与为每个突变体计算的结合能、几何改变和关键 YMDD 基序的变化进行比较。还将分析野生型的相应参数，计算出结合和未结合状态下 YMDD 基序的几何变化，以确定影响。分析药物在聚合酶催化位点上的结合将深入了解每个突变所赋予的耐药机制，更重要的是，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。该项目的第二阶段将通过与匹兹堡大学 Michael Parniak 实验室的合作来结合计算机 QMROI 研究和实验方法，该实验室致力于开发针对 RNH 位点的新型 RT 抑制剂。 [4]。QMROI方法将用于研究潜在的新抑制化合物的作用，指导此类化合物的设计并判断尚未观察到的突变的潜在影响。此类研究也将用于指导。 HIV-1 耐药性的定点突变研究 1. Ilina T，Parniak MA：HIV-1 逆转录酶抑制剂，药理学进展，56：121-167， 2008. 2. 何霞, 梅宇, 向宇, 张德文, 张建Z: 基于点突变的HIV-1逆转录酶对奈韦拉平耐药性的量子计算分析: 结构、功能与生物信息学, 61:423-432 , 2005. 3. Kohn W, Sham LJ: 非均匀介质中的量子密度振荡Electron Gas. Phys. Rev., 137(6A):1697-1705, 1965。 4. Himmel DM、Sarafinos SG、Dharmasina S、Parniak MA 等人：RNA 酶 H 抑制剂二羟基苯甲酰基萘腙的 HIV-1 逆转录酶结构绑定在新站点。ACS 化学生物学，1：702-711， 2006年。