Mapping epistatic interactions in molecular evolution of antibiotic resistance

项目总结 抗生素耐药性的演变是一个全球性的公共卫生问题。进化如何使抗生素 通过改变抗生素靶点使分子失效是临床和基础研究中的一个有趣的现象 科学视角。在致病菌中，只有少数几种药物靶标酶，如dna。 旋转酶、RNA聚合酶、脂肪酸合成酶和参与叶酸合成的酶。因此，a 从机制上理解这些酶的耐药突变在临床上是至关重要的 设计可以抑制耐药细菌的新药或药物变种。 在这个项目中，我们打算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MAP上位性相互作用。DHFR是一种普遍存在的酶，具有 在叶酸合成途径中起重要作用，并被用作抗菌、抗癌和 抗疟疾疗法。在细菌中，一种名为甲氧普林的抗生素竞争性地与DHFR结合并阻断其 催化活性。因此，产生抗药性或补偿催化作用降低的dhfr突变 抗dhfr突变体的活性被选择用于细菌生存。 我们将使用实验室进化实验来鉴定功能性dhfr突变和可重复性。 导致甲氧苄啶耐药性升高的遗传轨迹。我们将使用以下方法来表征这些突变 基于体外生化分析和深度测序的适合度测量用于计算上位性 DHFR突变之间的相互作用。我们将使用分子动力学和其他计算工具 和核磁共振波谱来揭示导致抗性的结构变化 和上位性相互作用。这些方法的结合提供了一个独特的机会来量化 评估导致甲氧苄氨嘧啶耐药的进化路径，并创建研究发现管道 蛋白质进化。通过加深对重要药物靶点的进化动力学的理解 酶，我们的计划将开发实验和计算工具，以研究蛋白质进化与 改善人类健康的终极目标。事实上，我们的初步分析表明，我们将能够 设计和测试可选择性抑制携带L28R的DHFR突变体的新型甲氧普林衍生物 替换，一种常见的协同DHFR突变。我们建议合成甲氧苄啶-二氢叶酸 杂化分子将同时具有DHF和甲氧苄啶分子的显著结构特征 用L28R替换选择性地抑制DHFR突变体。我们将在 为了量化突变的特定甲氧普林衍生物在阻止 抗药性的演变，并相应地制定新的战略，以更好地利用它。

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.