Using DNA-encoded Chemical Libraries to Develop Inhibitors of the MCR-1 Colistin Resistance Enzyme

使用 DNA 编码的化学文库开发 MCR-1 粘菌素抗性酶抑制剂

• 批准号: 10433324
• 负责人: Timothy Palzkill
• 金额: $ 24万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-25 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433324
• 关键词: Animal Model; Antibiotic Resistance; Antibiotics; Binding; Biochemical; C-terminal; Catalytic Domain; Charge; Chemicals; Clinical; Colistin; DNA; DNA sequencing; Effectiveness; Enterobacteriaceae; Enzymes; Escherichia coli; Future; Genes; Goals; Growth; Immobilization; In Vitro; Lead; Length; Libraries; Lipid A; Membrane; Minimum Inhibitory Concentration measurement; Monobactams; Multi-Drug Resistance; Mutation; N-terminal; Oligonucleotides; Pharmaceutical Chemistry; Pharmacology; Phosphatidylethanolamine; Plasmids; Polymyxins; Predisposition; Prevalence; Property; Proteins; Public Health; Resistance; Resort; Specificity; Structure-Activity Relationship; Testing; Transferase; Variant; beta-Lactamase; beta-Lactams; biophysical techniques; carbapenemase; colistin resistance; combinatorial; efficacy testing; experimental study; global health; inhibitor; inorganic phosphate; mutant; next generation; novel; pathogenic bacteria; phosphoethanolamine; resistance gene; small molecule; small molecule inhibitor; small molecule libraries

Using DNA-encoded Chemical Libraries to Develop Inhibitors of the MCR-1 Colistin Resistance Enzyme The increasing prevalence of bacterial pathogens resistant to multiple antibiotics is a serious threat to global health. The spread of multidrug-resistant Gram-negative bacterial pathogens is a particular concern. In this regard, the increased prevalence of carbapenemase enzymes such as the KPC and NDM b-lactamases has reduced the efficacy of b-lactam antibiotics. The limited treatment options has led to increased use of polymyxin antibiotics such as colistin. The recent emergence and spread of a plasmid-encoded, transferable resistance gene, mcr-1, is a cause for concern. The mcr-1 gene encodes an enzyme, MCR-1, that catalyzes the transfer of phosphoethanolamine (PEA) from phosphatidylethanolamine to the 1’ or 4’ phosphate of lipid A. The neutralization of the negative charge on lipid A reduces binding of the positively charged colistin, leading to resistance. The MCR-1 enzyme contains an N-terminal membrane domain and a soluble C-terminal catalytic domain. We have expressed and purified the full-length MCR-1 enzyme from E. coli and have shown it is active in vitro. The availability of the purified full-length enzyme provides the basis for a biochemical screen for small molecule inhibitors. DNA-encoded chemical libraries contain small molecules covalently attached to encoding DNA. These libraries have proven to be an efficient means of identifying small molecules that bind to a target protein. We screened a library containing >2 billion compounds against immobilized MCR-1 and identified potential hits by next-generation DNA sequencing. Several putative hit compounds were synthesized in the absence of the DNA tag and two of these molecules synergize with colistin to kill E. coli expressing the MCR-1 enzyme. Importantly, the compounds do not alter E. coli growth rate when used alone and do not synergize with colistin against a strain containing a catalytically inactive MCR-1 protein. We propose to validate these chemically unique compounds by showing that they bind and inhibit the MCR-1 enzyme. In addition, we will determine their spectrum of activity against a set of colistin-resistant clinical isolates containing the MCR- 1 enzyme as well as isolates containing the MCR-2, -3, -5, and -9 variant enzymes. Further, we will utilize medicinal chemistry approaches to determine structure-activity relationships and identify more potent derivatives of the compounds. Finally, we will screen new DNA-encoded chemical libraries to identify additional novel inhibitors. The end result of these studies will be validated, potent MCR-1 inhibitors that can be further developed with regard to pharmacological properties and efficacy in animal models in future studies. Our goal is to identify potent inhibitors of MCR-1 that will restore the effectiveness of current and future polymyxin antibiotics.

利用DNA编码的化学文库开发MCR-1粘菌素抗性抑制剂 酶 对多种抗生素耐药的细菌病原体日益流行，严重威胁着全球 健康。耐多药的革兰氏阴性细菌病原体的传播是一个特别令人担忧的问题。在这 在这方面，碳青霉烯酶，如KPC和NDM b-内酰胺酶的流行增加了 降低了β-内酰胺类抗生素的疗效。有限的治疗选择导致了更多的人使用 多粘菌素等抗生素。最近一种以质粒编码、可转移的病毒的出现和传播 耐药基因mcr-1令人担忧。Mcr-1基因编码一种酶，mcr-1，它催化 磷脂酰乙醇胺(PEA)向磷脂1‘或4’磷酸转移 A.脂类A上负电荷的中和减少了带正电荷的粘菌素的结合， 导致抵抗。MCR-1酶含有N-末端的膜区和可溶性的C-末端 催化域。我们已经从大肠杆菌中表达并纯化了全长的MCR-1酶，并展示了 它在体外具有活性。纯化的全长酶的可获得性为生化筛选提供了基础 用于小分子抑制剂。DNA编码的化学文库包含共价连接的小分子 编码DNA。这些文库已被证明是识别结合的小分子的有效手段 目标蛋白质的结合。我们筛选了一个包含20亿种化合物的文库，以对抗固定化的MCR-1和 通过下一代DNA测序确定了潜在的命中。合成了几种可能的热门化合物。 在没有DNA标签的情况下，其中两个分子与粘菌素协同作用杀死表达 MCR-1酶。重要的是，这些化合物在单独使用时不会改变大肠杆菌的生长速度，也不会 与粘菌素协同作用，对抗含有催化失活的MCR-1蛋白的菌株。我们建议验证 这些化合物在化学上是独特的，因为它们能结合和抑制MCR-1酶。此外，我们 将确定它们对一组含有MCR-的粘菌素耐药临床分离株的活性谱- 1酶以及含有MCR-2、-3、-5和-9变异酶的菌株。此外，我们将利用 确定构效关系和鉴定更有效的药物化学方法 化合物的衍生物。最后，我们将筛选新的DNA编码的化学库来识别 增加了新的抑制剂。这些研究的最终结果将是有效的MCR-1抑制剂，它可以 在未来的动物模型研究中，在药理特性和疗效方面有待进一步开发。 我们的目标是确定MCR-1的有效抑制剂，以恢复目前和未来的有效性 多粘菌素抗生素。