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：
9894816
负责人：
Erdal Toprak
金额：
$ 37.91万
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 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894816
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 Structure 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.

项目摘要抗生素耐药性的进化是全球公共卫生问题。进化如何呈现抗生素通过改变抗生素靶标的分子无效是临床和碱性的有趣现象科学观点。在致病细菌中，只有少数药物靶酶，例如DNA 回旋酶，RNA聚合酶，脂肪酸合成酶和参与叶酸合成的酶。因此，在这些酶中对抗性限制突变的机械理解在临床上至关重要设计可以抑制抗性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 DHFR突变之间的酶和绘制上皮相互作用。 DHFR是一种无处不在的酶在叶酸合成途径中的重要作用，并用作抗菌，抗癌和抗疟疾疗法。在细菌中，一种名为Trimethoprim的抗生素竞争与DHFR结合并阻止其催化活性。因此，赋予阻力或补偿催化性降低的DHFR突变选择抗性DHFR突变体的活性以进行细菌存活。我们将使用实验室进化实验来识别功能性DHFR突变和可重现遗传轨迹导致甲氧苄啶抗性升高。我们将通过使用这些突变来表征这些突变体外生化测定和基于深层的适应性测量值 DHFR突变之间的相互作用。我们将使用分子动力以及其他计算工具和核磁共振（NMR）光谱，以揭示导致电阻的结构变化和上毒互动。这些方法的组合为定量提供了独特的机会评估导致甲氧苄啶抗性的进化路径，并创建用于研究的发现管道蛋白质进化。通过对重要药物目标的进化动态有更深入的了解酶，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。确实，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，可以选择性地抑制携带L28R的DHFR突变体替代，一种常见和协同的DHFR突变。我们建议合成三甲氧苄啶二氢叶酸将具有DHF和甲氧苄啶分子的显着结构特征的混合分子选择性地抑制L28R替换的DHFR突变体。我们将在pan敏感的大肠杆菌中发展为了量化突变特异性甲氧苄啶衍生物在阻碍中的疗效阻力演变，并因此制定了更好地利用它的新策略。