Mapping epistatic interactions in molecular evolution of antibiotic resistance

绘制抗生素耐药性分子进化中的上位相互作用

基本信息

批准号：
10361439
负责人：
Erdal Toprak
金额：
$ 37.27万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-09 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10361439
关键词：
Affinity Algorithms Alleles Anti-Bacterial Agents Antibiotic Resistance Antibiotics Antimalarials Automobile Driving Bacteria Basic Science Biochemical Biological Assay Cells Cellular Metabolic Process Chemicals Clinical Clinical Sciences Competitive Binding Computational Biology Computer Analysis DNA Gyrase DNA-Directed RNA Polymerase Data Dihydrofolate Reductase Dihydrofolate Reductase Inhibitor Dose Drug Targeting Enzymes Escherichia coli Evolution Fatty Acids Folic Acid Free Energy Genes Genetic Genetic Epistasis Glutamates Goals Health Human Hybrids Hydrogen Bonding In Vitro Laboratories Lead Libraries Ligase Maps Measurement Methods Molecular Molecular Evolution Morbidity - disease rate Mutation NMR Spectroscopy Names Nuclear Magnetic Resonance Pathway interactions Pharmaceutical Preparations Phenotype Plasma Point Mutation Population Procedures Proteins Public Health Reproducibility Resistance Role Sampling Tail Testing Trimethoprim Trimethoprim Resistance Urine Variant anti-cancer bacterial resistance base clinically relevant combinatorial computerized tools cost deep sequencing design dihydrofolate drug-sensitive exhaustion experimental study fitness improved molecular dynamics mutant next generation sequencing novel novel therapeutics pathogenic bacteria preservation resistance mechanism synthetic construct tool

项目摘要

PROJECT SUMMARY Evolution of antibiotic resistance is a global public health problem. How evolution renders antibiotic molecules ineffective by altering antibiotic targets is an interesting phenomenon from both clinical and basic science perspectives. In pathogenic bacteria, there is only a handful of drug target enzymes such as DNA gyrases, RNA polymerases, fatty acid synthetases, and enzymes involved in folic acid synthesis. Therefore, a mechanistic understanding of resistance-conferring mutations in these enzymes is clinically critical for designing new drugs or drug variants that can inhibit resistant bacteria. In this project, we propose to study evolution of the Escherichia coli dihydrofolate reductase (DHFR) enzyme and map epistatic interactions between DHFR mutations. DHFR is a ubiquitous enzyme with an essential role in the folic acid synthesis pathway and is used as a drug target in antibacterial, anticancer, and antimalarial therapies. In bacteria, an antibiotic named trimethoprim competitively binds to DHFR and blocks its catalytic activity. Therefore, DHFR mutations that either confer resistance or compensate for reduced catalytic activity of resistant DHFR mutants are selected for bacterial survival. We will use laboratory evolution experiments to identify functional DHFR mutations and reproducible genetic trajectories leading to elevated trimethoprim resistance. We will characterize these mutations by using in vitro biochemical assays and deep-sequencing based fitness measurements for calculating epistatic interactions between DHFR mutations. We will use molecular dynamics along with other computational tools and nuclear magnetic resonance (NMR) spectroscopy to reveal structural changes responsible for resistance and epistatic interactions. The combination of these approaches presents a unique opportunity to quantitatively evaluate evolutionary paths leading to trimethoprim resistance and create a discovery pipeline for studying protein evolution. By creating a deeper understanding for the evolutionary dynamics of an important drug target enzyme, our proposal will develop experimental and computational tools for studying protein evolution with the ultimate goal of improving human health. Indeed, our preliminary analyses suggest that we will be able to design and test novel trimethoprim derivatives that can selectively inhibit DHFR mutants that carry the L28R replacement, a common and synergistic DHFR mutation. We propose to synthesize trimethoprim-Dihydrofolate hybrid molecules that will possess the salient structural features of both DHF and trimethoprim molecules selectively inhibit DHFR mutants with the L28R replacement. We will evolve pan sensitive E. coli strains in the morbidostat in order to quantify the efficacy of the mutant specific trimethoprim derivatives in impeding resistance evolution and accordingly develop new strategies for better use of it.

项目概要抗生素耐药性的演变是一个全球性的公共卫生问题。进化如何产生抗生素从临床和基础角度来看，通过改变抗生素靶点而无效的分子是一个有趣的现象科学观点。病原菌中只有DNA等少数药物靶点酶旋转酶、RNA聚合酶、脂肪酸合成酶和参与叶酸合成的酶。因此，一个对这些酶中赋予耐药性的突变的机制的理解对于临床至关重要设计可以抑制耐药细菌的新药或药物变体。在这个项目中，我们建议研究大肠杆菌二氢叶酸还原酶（DHFR）的进化酶并绘制 DHFR 突变之间的上位相互作用。 DHFR 是一种普遍存在的酶，在叶酸合成途径中发挥重要作用，并用作抗菌、抗癌和抗癌药物靶点抗疟疗法。在细菌中，一种名为甲氧苄啶的抗生素竞争性地与 DHFR 结合并阻断其催化活性。因此，DHFR 突变要么赋予抗性，要么补偿催化活性的降低选择抗性 DHFR 突变体的活性以保证细菌的存活。我们将使用实验室进化实验来鉴定功能性 DHFR 突变和可重复的突变导致甲氧苄啶耐药性升高的遗传轨迹。我们将通过使用来表征这些突变用于计算上位性的体外生化测定和基于深度测序的适应性测量 DHFR 突变之间的相互作用。我们将使用分子动力学以及其他计算工具和核磁共振 (NMR) 光谱揭示导致耐药性的结构变化和上位相互作用。这些方法的结合提供了一个独特的机会来定量分析评估导致甲氧苄啶耐药性的进化路径并创建用于研究的发现管道蛋白质进化。通过对重要药物靶标的进化动力学有更深入的了解酶，我们的建议将开发实验和计算工具，用于研究蛋白质进化改善人类健康的最终目标。事实上，我们的初步分析表明我们将能够设计和测试新型甲氧苄啶衍生物，可以选择性抑制携带 L28R 的 DHFR 突变体替代，一种常见且具有协同作用的 DHFR 突变。我们建议合成甲氧苄啶二氢叶酸杂化分子将具有 DHF 和甲氧苄啶分子的显着结构特征通过 L28R 替代选择性抑制 DHFR 突变体。我们将在以下环境中进化出泛敏感的大肠杆菌菌株 Morbidostat以量化突变体特定甲氧苄啶衍生物在阻碍中的功效抗性进化并相应地制定新策略以更好地利用它。