Evolution and inhibition of carbapenemase in beta-lactam resistance

碳青霉烯类抗生素曾经是最后的抗生素，不受内酰胺酶水解的影响，现在很容易被所谓的碳青霉烯酶失活，特别是在耐碳青霉烯类肠杆菌科(Cre，被CDC列为紧急威胁)中常见的以丝氨酸为基础的A类内酰胺酶KPC-2。碳青霉烯酶还威胁着目前正在开发的针对L、分枝杆菌D-转肽酶等的新碳青霉烯类抗生素的未来临床应用。然而，人们对KPC-2如何能够降解几乎所有的β-内酰胺类抗生素，以及如何通过耐药突变继续逃避新开发的抑制剂，如阿维巴坦，还知之甚少。此外，以NDM-1和VIM-2为代表的B类金属内酰胺酶已成为临床上常见的另一类有问题的碳青霉烯类酶，但几乎没有有效的抑制剂。通过基于结构的药物发现，我们已经确定了一系列基于磷酸的KPC-2抑制剂，其中最好的化合物表现出20 nM的结合亲和力和非常有希望的细胞活性。值得注意的是，这些化合物还表现出对金属碳青霉烯酶NDM-1和VIM-2的低M到高NM活性。对这些抑制剂和其他抑制剂的结构分析表明，碳青霉烯酶独特的活性部位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降解多种β-内酰胺类抗生素，但也使它们更容易受到不同小分子化学类型的抑制。在这项建议中，我们的目标是：1)利用基于结构的设计和合成、体外分析和动物模型，开发针对A类碳青霉烯酶特别是KPC-2的低至亚NM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)应用突变、X射线结晶学、核磁共振和MD模拟，探索支撑KPC-2‘S宽底物谱和独特的碳青霉烯酶活性的活性部位的静态和动态特征，以及研究包括我们自己的在内的新的抑制剂的抗药性发展。这些实验将为抗生素开发带来新的内酰胺酶抑制剂，同时加深对内酰胺酶催化和耐药性演变的理解，帮助指导未来的药物开发。

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.