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：
10598501
负责人：
Yu Chen
金额：
$ 73.04万
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/10598501
关键词：
Active Sites Affinity Animal Model Anti-Bacterial Agents Antibiotic Resistance Antibiotics Binding Biochemistry Carbapenems Catalysis Cells Chemicals Chemistry Clinic Clinical Collaborations Crystallography 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 Predisposition Process Production Property Protein Dynamics Research Personnel Resistance Resistance development Series Serine Structure Techniques Time X-Ray Crystallography analog animal efficacy bacterial resistance 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 molecular dynamics 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.

碳青霉烯是对β-内酰胺酶氢解的免疫曾经的最后一流的β-内酰胺抗生素，现在易于被所谓的碳青霉酶灭活，尤其是基于丝氨酸类的Aβ类Aclactamase KPC-2，通常在Carbapenem-Enterobactiac中常见于creSistant-Enterobacteac（Cretc），cree，cree，san an an an an an an an an an an and and cree，sane san and cree，cree，列表。碳青霉酶还威胁着目前针对分枝杆菌和其他人的L，D-转肽酶开发的新碳青霉烯的未来临床实用性。然而，众所周知，KPC-2如何能够水解几乎所有β-内酰胺抗生素，并通过抗性突变继续逃避新开发的抑制剂，例如阿维比巴坦。此外，由NDM-1和VIM-2代表的B类Metallo-β-β-内酰胺酶已成为诊所中经常观察到的另一个问题的碳二烯酶，但抑制剂很少。基于结构的药物发现，我们已经确定了一系列基于照片的KPC-2抑制剂，最佳化合物显示出20 nm的结合亲和力（Ki）和高度有希望的基于细胞的活性。值得注意的是，这些化合物还表现出对金属 - 核糖酶NDM-1和VIM-2的高NM活性低的M。对这些抑制剂和其他抑制剂的结构分析表明，碳青霉酶的独特活性位点特征似乎增强了它们与小分子结合的能力。这些特性使它们能够水解广泛的β-内酰胺抗生素，但也使它们更容易被潜水员小分子化学型抑制。在此提案中，我们的目标是：1）使用基于结构的设计和合成，体外分析和动物模型，对A级碳酸酶（尤其是KPC-2）的抑制剂（包括具有高亲和力）的双活性化合物，包括高亲和力，对A类抑制剂进行低至亚NM抑制剂； 2）应用诱变，X射线晶体学，NMR和MD模拟来探测静态和动态的活动位点特征，这些特征是KPC-2的广泛底物谱和独特的碳青霉烯酶活性，以及研究对现有和新抑制剂的抵抗力的发展。这些实验将导致新的ß-内酰胺酶抑制剂引导抗生素发育，同时对β-内酰胺酶催化和耐药的进化有更深入的了解，以帮助指导未来的药物发现。