Evolution and inhibition of carbapenemase in beta-lactam resistance

β-内酰胺耐药中碳青霉烯酶的进化和抑制

基本信息

批准号：
10385772
负责人：
Yu Chen
金额：
$ 73.85万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-06 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10385772
关键词：
Active Sites Affinity Animal Model Anti-Bacterial Agents Antibiotic Resistance Antibiotics Binding Biochemistry Carbapenems Catalysis Cells Centers for Disease Control and Prevention (U.S.)Chemicals Chemistry Clinic Clinical Collaborations Crystallization Cyclophosphamide Development Docking Enterobacteriaceae Enzymes Evolution FDA approved Future Genus Mycobacterium Goals Health Hydrolysis Hydrophobicity Immune In Vitro Infection Lactamase Lactams Lead Ligands Microbiology Modeling Modification Molecular Molecular Conformation Monobactams Multienzyme Complexes Mus Mutagenesis Mutation Penicillin-Binding Proteins Peptidyltransferase Pharmaceutical Chemistry Pharmaceutical Preparations Process Production Property Protein Dynamics Research Personnel Resistance Resistance development Resort Series Serine Structure Techniques Time X-Ray Crystallography analog animal efficacy bacterial resistance base beta-Lactam Resistance beta-Lactamase beta-Lactams carbapenem resistance carbapenem-resistant Enterobacteriaceae carbapenemase design drug discovery enzyme structure experience experimental study flexibility improved in vivo inhibitor mutant novel pathogenic bacteria phosphonate resistance mechanism resistance mutation scaffold simulation small molecule

项目摘要

Carbapenems, the once last-resort ß-lactam antibiotics immune to ß-lactamase hydrolysis, are now susceptible to inactivation by the so-called carbapenemases, especially the serine-based Class A ß-lactamase KPC-2 commonly found in carbapenem-resistant Enterobacteriaceae (CRE, listed as an urgent threat by CDC). Carbapenemases also threaten the future clinical utility of new carbapenems currently being developed against L,D-transpeptidases of mycobacteria and others. However, it is poorly understood how KPC-2 is able to hydrolyze nearly all ß-lactam antibiotics and continues to evade newly developed inhibitors, such as avibactam, via resistance mutations. Additionally, Class B metallo-ß-lactamases, represented by NDM-1 and VIM-2, have emerged as another problematic group of carbapenemases frequently observed in clinic, with yet few effective inhibitors. Through structure-based drug discovery, we have identified a series of phosphonate- based inhibitors of KPC-2, with the best compound displaying a binding affinity (Ki) of 20 nM and highly promising cell-based activities. Remarkably, these compounds also demonstrated low M to high nM activities against metallo-carbapenemases NDM-1 and VIM-2. Structural analysis of these inhibitors and others revealed that unique active site features of carbapenemases appear to enhance their ability to bind to small molecules. These properties enable them to hydrolyze a wide range of ß-lactam antibiotics but also make them more prone to inhibition by diverse small molecule chemotypes. In this proposal, we aim to: 1) develop low to sub- nM inhibitors against Class A carbapenemases particularly KPC-2, including dual-activity compounds with high affinity for metallo-carbapenemases as well, using structure-based design and synthesis, in vitro analysis and animal models; 2) apply mutagenesis, X-ray crystallography, NMR and MD simulation to probe the active site features, both static and dynamic, that underlie KPC-2’s broad substrate profile and unique carbapenemase activity, as well as to investigate the development of resistance against existing and new inhibitors including our own. These experiments will result in new ß-lactamase inhibitor leads for antibiotic development, while providing a deeper understanding of ß-lactamase catalysis and the evolution of resistance, to help guide future drug discovery.

碳青霉烯类曾经是对 β-内酰胺酶水解免疫的最后手段 β-内酰胺抗生素，现在很容易被所谓的碳青霉烯酶灭活，特别是常见于碳青霉烯类耐药肠杆菌科细菌中常见的基于丝氨酸的 A 类 β-内酰胺酶 KPC-2 （CRE，被 CDC 列为紧急威胁）也威胁着目前正在开发的新型碳青霉烯类的未来临床应用。然而，人们对KPC-2如何能够水解几乎所有β-内酰胺抗生素并通过耐药突变继续逃避新开发的抑制剂（例如avibactam）知之甚少。以 NDM-1 和 VIM-2 为代表的 B 金属-β-内酰胺酶已成为临床上常见的另一组有问题的碳青霉烯酶，但有效的方法很少。通过基于结构的药物发现，我们高度鉴定了一系列基于磷酸盐的 KPC-2 抑制剂，其中最好的化合物显示出 20 nM 的结合亲和力 (Ki) 和令人瞩目的细胞活性。还表现出针对金属碳青霉烯酶 NDM-1 和 VIM-2 的低 M 至高 nM 活性。这些抑制剂和其他抑制剂的结构分析揭示了碳青霉烯酶的独特活性位点特征。这些特性似乎增强了它们与小分子结合的能力，使它们能够水解多种β-内酰胺抗生素，但也使它们更容易受到不同小分子化学类型的抑制。在本提案中，我们的目标是：1）开发。使用基于结构的设计和合成、体外分析和动物模型，针对 A 类碳青霉烯酶（特别是 KPC-2）的低至亚纳米级抑制剂，包括对金属碳青霉烯酶具有高亲和力的双活性化合物； 2) 应用诱变、X射线晶体学、NMR和MD模拟来探测活性位点特征（静态和动态），这些特征是KPC-2广泛的底物特征和独特的碳青霉烯酶活性的基础，并研究对现有的耐药性的发展这些实验将为抗生素开发带来新的β-内酰胺酶抑制剂先导化合物，同时提供对β-内酰胺酶催化和耐药性进化的更深入了解，以帮助指导未来的药物。发现。