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

• 批准号: 10735464
• 负责人: Erdal Toprak
• 金额: $ 38.86万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-09 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735464
• 关键词: Address; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Biochemical; Biological Assay; Biological Sciences; Carbapenems; Cephalosporinase; Cephalosporins; Chemicals; Clinic; Clinical; Communicable Diseases; Cyclophosphamide; DNA Gyrase; Dihydrofolate Reductase; Dihydrofolate Reductase Inhibitor; Drug Targeting; Engineering; Enzymes; Escherichia coli; Evolution; FDA approved; Folic Acid; Future; Genes; Genetic; Genetic Epistasis; Genotype; Goals; Gram-Negative Bacteria; Health; Hydrolysis; In Vitro; Induced Mutation; Interdisciplinary Study; Lactamase; Lactams; Libraries; Life; Maps; Mathematics; Metabolism; Modeling; Modification; Molecular Evolution; Monobactams; Mutagenesis; Mutation; Names; Non-Steroidal Anti-Inflammatory Agents; Operative Surgical Procedures; Pathway interactions; Patient-Focused Outcomes; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Procedures; Prodrugs; Public Health; Research; Resistance; Role; Shapes; Site; Testing; Time; Trimethoprim; Trimethoprim Resistance; Variant; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; design; effective therapy; fitness; inhibitor; innovation; mutant; novel; novel drug class; programs; resistance mechanism; small molecule; therapeutic target; tool; treatment strategy

PROJECT SUMMARY Antibiotic resistance is one of the most serious public health challenges of our time. In this proposal, we focus on two common antibiotic resistance mechanisms that are: (i) alteration of drug target enzymes and (ii) modification of antibiotic molecules. Despite recent advances in biological sciences, clinically accessible antibiotics can still target only a handful of enzymes, such as DNA gyrases and dihydrofolate reductase (DHFR). Hence, it is important to better understand molecular evolution of these enzymes and accordingly develop effective drugs that target these enzymes without exacerbating the resistance problem. Similarly, modification of -lactam antibiotics through hydrolysis by -lactamases is currently the most concerning antibiotic resistance mechanism because -lactams account for nearly seventy percent of antibiotics currently used in clinics. To address this important health problem, we propose an innovative research plan to study evolution of the DHFR enzyme, and develop mutant-specific competitive and allosteric DHFR inhibitors to select against resistance- conferring DHFR mutations. Finally, to address the -lactam resistance problem, we will engineer a novel class of molecules that we named “-lactamase traps” (or BLTs) to select against -lactamase producing bacteria. Our first aim is to map epistatic interactions that shape DHFR evolution under antibiotic selection. DHFR is a ubiquitous enzyme with a central role in metabolism. We have previously shown that E. coli DHFR accumulates 3 to 5 resistance conferring mutations following a quasi-deterministic order, as a result of strong epistatic interactions between DHFR mutations. We will quantitatively map all epistatic interactions in the DHFR fitness landscape by using state-of-the-art genetic tools and the Hierarchical Model, a mathematical toolbox we developed to efficiently explore the fitness space and identify epistatic interactions between mutations. Our second goal is to engineer competitive and allosteric mutant-specific DHFR inhibitors. We have previously developed an L28R-specific competitive trimethoprim derivative (4’-dTMP) that impeded evolution of resistance by selecting against the L28R mutation. Following a similar procedure, we will engineer new competitive DHFR inhibitors that will target resistance-conferring mutations on the D27, W30, I94, and F153 residues. Similarly, we will identify allosteric inhibitors that can target a druggable DHFR cryptic site we discovered. This site has limited drug accessibility for the wild-type DHFR but always remains open for the D27E, I94L, and F153S variants of DHFR. We already showed that proglumetacin, a commonly used NSAID drug, allosterically slows down DHFR activity. Our third goal is to engineer a novel class of molecules that select against -lactamase genes. We will develop a novel class of molecules that we call BLTs to eliminate -lactamase producing Gram-negative bacteria. BLT molecules are inactive in their native form but once activated by -lactamases, turn into potent antibiotics. We have already designed and synthesized a cephalosporin-based BLT molecule that is activated by the CTX-M cephalosporinase. We will generate a library of cephalosporin- and carbapenem-based BLT molecules.

项目摘要 抗生素耐药性是我们时代最严重的公共卫生挑战之一。在此提案中，我们集中于 在两种常见的抗生素抗性机制上是：（i）药物靶酶的改变和（ii） 抗生素分子的修饰。尽管生物科学的最新进展，但在临床上可访问 抗生素仍然只能靶向少数酶，例如DNA回旋酶和二氢叶酸还原酶（DHFR）。 因此，重要的是要更好地了解这些酶的分子进化并因此发展 靶向这些酶的有效药物而不会加剧耐药性问题。同样，修改 -内酰胺酶通过-内乳酶水解的抗生素是目前最令人抗性的抗生素耐药性 机制是因为-内酰胺占诊所目前使用的抗生素的近70％。到 解决这个重要的健康问题，我们提出了一项创新的研究计划来研究DHFR的演变 酶，并产生突变特异性竞争和变构DHFR抑制剂，以选择抗性 - 赋予DHFR突变。最后，为了解决-lactam的抵抗问题，我们将设计一个新颖的课程 我们命名为“-内酰胺酶陷阱”（或blts）的分子以选择-内酰胺酶产生细菌。 我们的第一个目的是绘制在抗生素选择下塑造DHFR进化的上皮相互作用。 DHFR是一个 无处不在的酶在代谢中具有中心作用。我们以前已经表明大肠杆菌DHFR积累 由于强烈的认识，准确确定秩序后的3至5抗性会议突变 DHFR突变之间的相互作用。我们将定量映射DHFR健身中的所有上皮相互作用 景观通过使用最先进的遗传工具和分层模型，这是一种数学工具箱 开发的旨在有效地探索健身空间并确定突变之间的上皮相互作用。我们的 第二个目标是设计具有竞争力和变构突变特异性DHFR抑制剂。我们以前有 开发了L28R特异性竞争性甲氧苄啶衍生物（4'-DTMP），阻碍了电阻的演变 通过选择L28R突变。按照类似的程序，我们将设计新的竞争性DHFR 将在D27，W30，I94和F153残留物上靶向抗性的突变的抑制剂。同样，我们 将确定可以针对我们发现的可吸毒DHFR加密位点的变构抑制剂。该网站有限 野生型DHFR的药物可访问性，但始终为D27E，I94L和F153S变体开放 DHFR。我们已经表明，proglumetacin是一种常用的NSAID药物，变构减慢了DHFR 活动。我们的第三个目标是设计一种针对-内酰胺酶基因的新型分子。我们将 开发出一种新型的分子，我们称之为BLT，以消除产生革兰氏阴性细菌的-内酰胺酶。 BLT分子以其天然形式不活跃，但曾经被-内乳糖酶激活，会变成潜在的抗生素。 我们已经设计和合成了基于头孢菌素的BLT分子，该分子被激活 CTX-M头孢菌素酶。我们将生成一个基于头孢菌素和碳青霉烯的BLT分子的库。