De novo design of small-molecule-binding proteins

小分子结合蛋白的从头设计

基本信息

批准号：
10604467
负责人：
Nicholas Polizzi
金额：
$ 24.9万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10604467
关键词：
Achievement Affinity Agreement Algorithm Design Amino Acid Sequence Amino Acids Amino Acyl-tRNA Synthetases Anticoagulants Antidotes Antithrombin III Award Binding Binding Proteins Binding Sites Charge Clinical Coagulation Process Collaborations Complement Complex Computer Models Computing Methodologies Crystallization Databases Development Drug Delivery Systems Enzymes Event Evolution Fibrinolytic Agents Geometry Goals Health Heterogeneity Human Hydrogen Bonding Hydrophobicity Individual Knowledge Laboratories Learning Libraries Life Ligands Ligase Mammalian Cell Mediating Medical Methods Modernization Molecular Biology Molecular Conformation Molecular Probes Motivation Mutagenesis Nature Pharmaceutical Preparations Pharmacologic Substance Phosphoserine Physicians Play Porphyrins Post-Translational Protein Processing Principal Investigator Process Protein Engineering Protein Structure Databases Proteins Publications Recording of previous events Research Research Training Resolution Roentgen Rays Role Sampling Schedule Scientist Set protein Specificity Structure System Temperature Testing Time Training Variant Vertebral column Water Work X-Ray Crystallography base clinical application clinically relevant combinatorial data repository delivery vehicle design functional group infancy insight meetings model design molecular dynamics molecular recognition mutant novel phosphohistidine porphyrin a professor programs protein data bank protein folding protein function protein protein interaction pyrrolysine screening sensor small molecule success tyrosine O-sulfate unnatural amino acids water sampling

项目摘要

PROJECT SUMMARY Protein-ligand binding events underlay all life processes. Protein design tests and extends our knowledge of protein folding and function through the creation of proteins from scratch. This proposal aims to develop a computational method for the design of proteins that bind to any small molecule with high affinity and selectivity. The state-of-the-art in ligand-binding protein design critically relies on random experimental optimization and screening. If we truly understand how proteins bind small molecules, we should be able to go directly from computer models to tight binders. The hypothesis that drives this proposal is that proteins use a vast but now enumerable number of molecular interaction motifs combinatorially throughout evolution to create the binding sites of modern-day proteins. Computational methods will be employed to uncover this set of interactions in the large database of protein structures available in the protein databank (PDB). Binding sites will be designed by sampling motifs for all functional groups of a ligand onto a protein backbone. We call this design method Convergent Motifs for Binding Sites (COMBS). COMBS was used to design ABLER, the first ligand-binding protein designed from scratch to bind its target ligand—the antithrombotic drug apixaban—with an unprecedentedly high affinity, without experimental optimization of sequence. ABLER has potential clinical relevance as an anti-clotting antidote, although that is outside the scope of the proposal. High-resolution crystal structures of ABLER agree with the design model, both in overall topology and the intended molecular interactions with the ligand. Aim 1 of this proposal focuses on designing variants of ABLER to increase affinity and probe the molecular bases for the observed drug-protein interactions. Aim 2 focuses on the role of water in ligand-binding protein design, motivated by the water-mediated protein-ligand interaction found in the crystal structure. In this Aim, I will curate a database of water-protein interactions from the PDB and use these to sample water-mediated protein-ligand interactions during design. I will also learn to use explicit-water molecular dynamics simulations to critically assess the roles of water in binding. Aim 3 uses COMBS to redesign the binding site of pyrrolysine tRNA synthetase for incorporation of charged unnatural amino acids (such as sulfotyrosine) into mammalian cells, since laboratory evolution and library screens for this goal have so far been unsuccessful. These aims will augment my training in molecular biology, computational protein design, and protein structural characterization (X-ray crystallography and NMR). The K99 portion of this work in the DeGrado lab will expose me to all aspects of the scientific process, from inception to publication. Bill is a world expert in protein design, and his insight is critical to the success of the project. At UCSF, I will gain much through my regularly scheduled meetings with Ethan Weiss, who brings the perspective of a physician scientist with a long history of antithrombotic research and clinical applications. My collaboration with UCSF professor Lei Wang will expose me to the field of unnatural amino acid incorporation and will be critical for applying COMBS to the most impactful targets for mimics of post-translational modifications. The research and training proposed herein will greatly complement my current skillset and background, which will be essential to my research program as I transition into an independent principal investigator.

项目摘要蛋白质结合事件构成了所有生命过程。蛋白质设计测试并扩展了我们对蛋白质折叠和通过从头开始创建蛋白质的功能。该建议旨在发展用于设计与任何具有高亲和力的小分子的蛋白质设计的计算方法选择性。配体结合蛋白设计的最先进的依赖于随机实验优化和筛选。如果我们真正了解蛋白质如何结合小分子，那么我们应该能够去直接从计算机型号到紧密的粘合剂。驱动该提议的假设是蛋白质使用庞大但现在枚举数量的分子相互作用图案在整个演变中结合起来，以创建现代蛋白质的结合位点。计算方法将被雇用以发现这组蛋白质结构的大数据库中的相互作用可在蛋白质数据库（PDB）中提供。绑定位点将通过对所有配体官能团在蛋白质主链上的所有功能组进行采样基序设计。我们称之为设计方法收敛基序（梳子）。梳子被用来设计泡沫，第一个配体结合蛋白从头开始设计，以结合其靶配体（抗强化药物apixaban）前所未有的高亲和力，没有序列实验优化。 Abler具有潜在的临床尽管这超出了提案的范围，但相关性是一种反封闭解毒剂。高分辨率晶体 ABLER的结构在整体拓扑和预期分子方面都与设计模型一致与配体的相互作用。该提案的目标1着重于设计ABLER的变体以增加亲和力并探测观察到的药物蛋白相互作用的分子碱基。 AIM 2专注于水中的作用配体结合蛋白设计，由在晶体中发现的水介导的蛋白质配体相互作用成熟结构。在此目标中，我将策划一个来自PDB的水 - 蛋白质相互作用数据库，并将其使用在设计过程中，样品水介导的蛋白质 - 配体相互作用。我还将学会使用显式水分子动力学模拟，以批判性地评估水在结合中的作用。 AIM 3使用梳子重新设计用于带电的非天然氨基酸保险的吡咯氨氨酸TRNA合成酶的结合位点（例如硫酪氨酸）进入哺乳动物细胞，因为该目标的实验室进化和图书馆筛选已经是迄今为止不成功。这些目标将增加我在分子生物学，计算蛋白设计和蛋白质结构表征（X射线晶体学和NMR）。这项工作的K99部分 Degrado Lab将使我了解从成立到出版的科学过程的各个方面。比尔是一个世界蛋白质设计方面的专家及其见解对于该项目的成功至关重要。在UCSF，我将获得很多收获我定期与Ethan Weiss举行的会议，他带来了一位物理科学家的观点抗血栓研究和临床应用的悠久历史。我与UCSF Lei教授的合作王会让我接触到不自然的氨基酸掺入的领域，对于施用梳子至关重要对于模仿翻译后修饰的最有影响力的目标。提出的研究和培训这里将大大完成我当前的技能和背景，这对我的研究至关重要当我过渡到独立的首席研究员时，计划。