Evolution and inhibition of carbapenemase in beta-lactam resistance

碳青霉烯类，曾经是对β-内酰胺酶水解免疫的最后手段的β-内酰胺抗生素，现在容易被所谓的碳青霉烯酶灭活，特别是通常在碳青霉烯耐药肠杆菌科（CRE，被CDC列为紧急威胁）中发现的基于丝氨酸的A类β-内酰胺酶KPC-2。碳青霉烯酶还威胁到目前正在开发的针对分枝杆菌和其他分枝杆菌的L，D-转肽酶的新型碳青霉烯类的未来临床用途。然而，人们对KPC-2如何能够水解几乎所有的β-内酰胺抗生素并通过耐药性突变继续逃避新开发的抑制剂（如阿维巴坦）知之甚少。此外，B类金属β-内酰胺酶，以NDM-1和Vim-2为代表，已成为临床上经常观察到的另一组有问题的碳青霉烯酶，但几乎没有有效的抑制剂.通过基于结构的药物发现，我们已经鉴定了一系列基于膦酸酯的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）和非常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碳青霉烯酶NDM-1和Vim-2的低nM至高nM活性。对这些抑制剂和其他抑制剂的结构分析显示，碳青霉烯酶独特的活性位点特征似乎增强了其与小分子结合的能力。这些性质使它们能够水解广泛的β-内酰胺抗生素，但也使它们更容易受到不同小分子化学型的抑制。1）使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也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，包括静态和动态，这是KPC-2广泛的底物谱和独特的碳青霉烯酶活性的基础，以及研究对现有和新的抑制剂（包括我们自己的抑制剂）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，同时提供对β-内酰胺酶催化和耐药性演变的更深入了解，以帮助指导未来的药物发现。

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.