Design and Experimental Testing of New Docking Methods

新对接方法的设计和实验测试

• 批准号: 8816927
• 负责人: Brian K Shoichet
• 金额: $ 33.68万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8816927
• 关键词: Address; Adopted; Affinity; Algorithms; Area; Aspartate; Binding; Biological; Biological Models; Biology; Charge; Clinical; Complement; Complex; Crystallography; Cytochromes; Development; Docking; Drug Targeting; Engineering; Ensure; Enzymatic Biochemistry; Enzymes; Equilibrium; Experimental Designs; Experimental Models; G Protein-Coupled Receptor Genes; Goals; Health; Hydrogen Bonding; Lactamase; Libraries; Ligands; Methods; Modeling; Molecular; Molecular Conformation; Muramidase; Opioid; Opioid Receptor; Peroxidases; Pharmaceutical Preparations; Pharmacologic Substance; Phosphotransferases; Physics; Proteins; Renaissance; Resolution; Role; Sampling; Site; Solvents; Structure; System; Techniques; Testing; Variant; Water; Weight; Work; adduct; flexibility; improved; inhibitor/antagonist; meetings; method development; mutant; programs; prospective; receptor; screening; small molecule; success; theories

DESCRIPTION (provided by applicant): Molecular docking is now widely used for ligand discovery. The technique makes many approximations and though it has had noteworthy successes, it is neither fully reliable nor do we understand why it fails when it does. Here we develop new docking methods that are tested in six model protein cavities engineered for docking. Each is small (150 to 200 Å3), buried from solvent, and dominated by a single term: from a fully hydrophobic cavity, to the same cavity with a single hydrogen-bond acceptor, to a cavity dominated by a single aspartate, and intermediates between. In these cavities particular interactions may be isolated and failed predictions are as illuminating as confirmed ones. Against each cavity we screen a library of 500,000 small molecules, testing predicted ligands for affinity (e.g., by ITC) and geometric fidelity, by x-ray. These studies are extended to several tru biological targets to ensure relevance. The specific aims are: 1. Development of new docking algorithms. We match methods development with experimental testing, focusing on areas where pragmatic improvement may be anticipated. A. Sampling receptor flexibility while weighting introduced conformational strain, exploiting crystallographic occupancies from new refinement techniques (tested in 2.A). B. Modeling ordered water by exploiting Inhomogenous Solvation Theory tested in (tested in 2.B) and also against a more complicated drug target, the μ-opioid GPCR. C. Exploring Higher-levels of ligand partial charges and solvation energies to improve the balance between apolar, polar and ionic energies in docking (tested in 2.C). D. A covalent docking method, seeking adduct formation between electrophilic ligand and a protein nucleophile (tested in 2.D). 2. Testing the new methods in model systems. A. Receptor flexibility is tested in prospective docking screens against a cavity in Cytochrome C Peroxidase (CCP) that bares a flexible loop whose multiple conformations may be observed and weighted from an apo-structure. B. The IST method for Ordered waters is tested against the six waters observed in cavity W191G and the eight in W191G/D192-3, investigating our ability to prospectively predict new ligands that exploit these waters. This work is extended to a more complicated target, the μ-opioid receptor. C. The balance among polar, apolar, and ionic terms is tested vs pairs of cavities differing by single polar substitutions (lysozyme L99A--> L99A/M102H; CCP W191G-->W191G/D235N). D. Covalent docking is prospectively tested against two nucleophilic enzymes, β-lactamase and RSK2 kinase, combining enzymology and x-ray crystallography to evaluate the binding of reversible covalent inhibitors predicted by the new method.

描述(申请人提供)：分子对接现在被广泛用于配基发现。这项技术进行了许多近似，尽管它取得了值得注意的成功，但它既不完全可靠，我们也不理解它为什么会失败。在这里，我们开发了新的对接方法，并在为对接而设计的六个模型蛋白质腔中进行了测试。每一个都很小(150至200ä3)，隐藏在溶剂中，并由一个术语主导：从完全疏水的空腔，到具有单一氢键受体的相同空腔，再到由单一天冬氨酸主导的空腔，以及中间体之间。在这些空穴中，特定的相互作用可能是孤立的，失败的预测与确认的预测一样具有启发性。对于每个空洞，我们筛选一个包含500,000个小分子的文库，通过X射线测试预测的配体的亲和力(例如，通过ITC)和几何保真度。这些研究扩展到几个TRU生物目标，以确保相关性。具体目标是：1.开发新的对接算法。我们将方法开发与实验测试相匹配，专注于可能预期实际改进的领域。A.在称重的同时采样受体的灵活性，引入构象应变，利用新的提纯技术的晶体占有率(在2.A中进行了测试)。B.利用非均相溶剂化理论对有序水进行建模(在2.B中进行了测试)，并针对更复杂的药物靶标μ-阿片类药物GPCR进行了测试。探索更高水平的配基部分电荷和溶剂化能，以改善对接中非极、极性和离子能量之间的平衡(在2.C下测试)。D.一种共价对接方法，寻求亲电配体和蛋白质亲核试剂之间的加合物形成(在2.D中测试)。2.在模型系统中对新方法进行测试。答：受体的灵活性在预期的对接屏幕上进行测试，对照细胞色素C过氧化物酶(CCP)中的一个空腔，该空腔暴露出一个灵活的环，其多重构象可以从脱辅基结构中观察和加权。B.有序水域的IST方法与在W191G腔中观察到的6个水域和W191G/D192-3中的8个水域进行了测试，调查了我们前瞻性预测利用这些水域的新配体的能力。这项工作扩展到了一个更复杂的靶点--μ阿片受体。C.通过单极性取代不同的空穴对(溶菌酶L99A--&GT；L99A/M102H；CCP W191G--&GT；W191G/D235N)，测试了极性项、非极性项和离子项之间的平衡。D.共价对接是针对两种亲核酶，β-内酰胺酶和RSK2激酶进行的前瞻性测试，结合酶学和X射线结晶学来评估新方法预测的可逆共价抑制剂的结合。