Evolution and inhibition of carbapenemase in beta-lactam resistance

碳青霉烯类，曾经是对ß-内酰胺酶水解免疫的最后一种ß-内酰胺类抗生素，现在容易被所谓的碳青霉烯类酶失活，尤其是在碳青霉烯类耐药肠杆菌科（CRE，被CDC列为紧急威胁）中常见的丝氨酸类A ß-内酰胺酶KPC-2。碳青霉烯酶也威胁到目前正在开发的针对分枝杆菌L、d转肽酶等的新型碳青霉烯类药物的临床应用前景。然而，KPC-2如何能够水解几乎所有的ß-内酰胺类抗生素，并通过耐药突变继续逃避新开发的抑制剂，如阿维巴坦，目前尚不清楚。此外，以NDM-1和VIM-2为代表的B类金属ß-内酰胺酶是临床常见的另一类问题型碳青霉烯酶，有效抑制剂尚少。通过基于结构的药物发现，我们已经确定了一系列基于膦酸盐的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）和极有希望的细胞活性。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至高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多种ß-内酰胺类抗生素，但也使它们更容易受到各种小分子化学型的抑制。在本研究中，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，开发低至亚纳米级的抗A类碳青霉烯酶抑制剂，特别是KPC-2，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KPC-2广泛的底物特征和独特的碳青霉烯酶活性的活性位点特征（静态和动态），并研究对现有和新的抑制剂（包括我们自己的抑制剂）的抗性发展。这些实验将为抗生素的开发提供新的ß-内酰胺酶抑制剂先导物，同时对ß-内酰胺酶的催化作用和耐药性的演变提供更深入的了解，有助于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.