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这个时代最严重的公共卫生挑战之一。在本提案中，我们将重点 两种常见的抗生素耐药机制是：（i）药物靶酶的改变和（ii） 抗生素分子的修饰。尽管生物科学取得了最新进展，但临床上可获得的 抗生素仍然只能针对少数酶，如DNA促旋酶和二氢叶酸还原酶（DHFR）。 因此，更好地理解这些酶的分子进化并相应地开发它们是重要的。 针对这些酶而不加剧耐药性问题的有效药物。同样，修改 β-内酰胺类抗生素通过β-内酰胺酶水解产生的耐药性是目前最受关注的抗生素耐药性 这是因为β-内酰胺类抗生素占目前临床使用的抗生素的近70%。到 为了解决这一重要的健康问题，我们提出了一个创新的研究计划来研究DHFR的进化 酶，并开发多能特异性竞争性和变构DHFR抑制剂，以选择抵抗- 赋予DHFR突变。最后，为了解决β-内酰胺耐药性问题，我们将设计一个新的类， 我们命名为"β-内酰胺酶陷阱"（或BLT）的分子，以选择对抗产生β-内酰胺酶的细菌。 我们的第一个目标是绘制抗生素选择下DHFR进化的上位相互作用。DHFR是一个 在新陈代谢中起中心作用的普遍存在的酶。我们以前已经证明，E。大肠杆菌DHFR积累 由于强上位性，3至5个耐药突变遵循准确定性顺序， DHFR突变之间的相互作用。我们将定量映射DHFR适应度中的所有上位相互作用 通过使用最先进的遗传工具和层次模型，一个数学工具箱， 开发用于有效地探索适应度空间并识别突变之间的上位相互作用。我们 第二个目标是设计竞争性和变构性的突变体特异性DHFR抑制剂。我们先前已经 开发了一种L28R特异性竞争性甲氧苄啶衍生物（4'-dTMP）， 通过对L28R突变进行选择。按照类似的程序，我们将设计新的竞争DHFR 靶向D27、W30、I94和F153残基上的抗性赋予突变的抑制剂。同样我们 将鉴定出能够靶向我们发现的可药用DHFR隐蔽位点的变构抑制剂。本网站有限 野生型DHFR的药物可及性，但对于DHFR的D27E、I94L和F153S变体始终保持开放。 DHFR。我们已经证明了葡美辛，一种常用的非甾体抗炎药， 活动我们的第三个目标是设计出一种新型的分子，可以选择β-内酰胺酶基因。我们将 开发了一类新的分子，我们称之为BLT，以消除产生β-内酰胺酶的革兰氏阴性细菌。 BLT分子在其天然形式下是无活性的，但一旦被β-内酰胺酶激活，就变成有效的抗生素。 我们已经设计并合成了一种基于头孢菌素的BLT分子， CTX-M头孢菌素酶。我们将生成基于头孢菌素和碳青霉烯的BLT分子库。