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

基本信息

批准号：
10613563
负责人：
Timothy Palzkill
金额：
$ 20万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-25 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10613563
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 Lactamase Lead Length Libraries Lipid A Membrane Minimum Inhibitory Concentration measurement Monobactams Multi-Drug Resistance Mutation N-terminal Oligonucleotides Pharmaceutical Chemistry Phosphatidylethanolamine Plasmids Polymyxins Predisposition Prevalence Property Proteins Public Health Resistance Specificity Structure-Activity Relationship Testing Transferase Variant biophysical techniques carbapenemase colistin resistance combinatorial efficacy testing experimental study global health inhibitor inorganic phosphate mutant next generation novel pathogenic bacteria pharmacologic phosphoethanolamine resistance gene small molecule small molecule inhibitor small molecule libraries synergism

项目摘要

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.

使用 DNA 编码的化学文库开发 MCR-1 粘菌素耐药性抑制剂酶对多种抗生素产生耐药性的细菌病原体的日益流行对全球造成严重威胁多重耐药革兰氏阴性细菌病原体的传播是一个特别值得关注的问题。考虑到碳青霉烯酶（例如 KPC 和 NDM β-内酰胺酶）的流行率增加，有限的治疗选择导致了β-内酰胺抗生素的使用增加。多粘菌素抗生素，例如粘菌素，最近出现并传播了一种质粒编码的、可转移的。抗性基因 mcr-1 令人担忧 mcr-1 基因编码一种催化酶 MCR-1。磷酸乙醇胺 (PEA) 从磷脂酰乙醇胺转移至脂质的 1' 或 4' 磷酸酯 A. 脂质 A 上的负电荷的中和减少了带正电荷的粘菌素的结合， MCR-1 酶含有 N 端膜结构域和可溶性 C 端。我们已经从大肠杆菌中表达并纯化了全长 MCR-1 酶，并已证明。纯化的全长酶的可用性为生化筛选提供了基础。对于小分子抑制剂，DNA 编码的化学文库包含共价连接的小分子。这些编码 DNA 的文库已被证明是识别结合小分子的有效方法。我们筛选了一个包含超过 20 亿个针对固定化 MCR-1 的化合物的文库，通过下一代 DNA 测序鉴定了潜在的命中化合物，并合成了几种假定的命中化合物。在没有 DNA 标签的情况下，其中两个分子与粘菌素协同作用，杀死表达重要的是，这些化合物单独使用时不会改变大肠杆菌的生长速度。与粘菌素协同对抗含有催化失活的 MCR-1 蛋白的菌株我们建议进行验证。此外，我们还发现这些化学上独特的化合物能够结合并抑制 MCR-1 酶。将确定其针对一组含有 MCR- 的耐粘菌素临床分离株的活性谱 1 酶以及含有 MCR-2、-3、-5 和 -9 变体酶的分离物。药物化学方法确定结构-活性关系并识别更有效的最后，我们将筛选新的 DNA 编码化学库来鉴定。这些研究的最终结果将是有效的 MCR-1 抑制剂。在未来的研究中，将进一步开发动物模型的药理学特性和功效。我们的目标是确定 MCR-1 的有效抑制剂，以恢复当前和未来的有效性多粘菌素抗生素。