Department of Biology, University of Texas at Arlington, Arlington, TX, USA.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
J Antimicrob Chemother. 2020 Oct 1;75(10):2843-2851. doi: 10.1093/jac/dkaa242.
Metallo-β-lactamases (MBLs) are an emerging class of antimicrobial resistance enzymes that degrade β-lactam antibiotics, including last-resort carbapenems. Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are increasingly prevalent, but treatment options are limited. While several serine-dependent β-lactamase inhibitors are formulated with commonly prescribed β-lactams, no MBL inhibitors are currently approved for combinatorial therapies. New compounds that target MBLs to restore carbapenem activity against CPE are therefore urgently needed. Herein we identified and characterized novel synthetic peptide inhibitors that bound to and inhibited NDM-1, which is an emerging β-lactam resistance mechanism in CPE.
We leveraged Surface Localized Antimicrobial displaY (SLAY) to identify and characterize peptides that inhibit NDM-1, which is a primary carbapenem resistance mechanism in CPE. Lead inhibitor sequences were chemically synthesized and MBCs and MICs were calculated in the presence/absence of carbapenems. Kinetic analysis with recombinant NDM-1 and select peptides tested direct binding and supported NDM-1 inhibitor mechanisms of action. Inhibitors were also tested for cytotoxicity.
We identified approximately 1700 sequences that potentiated carbapenem-dependent killing against NDM-1 Escherichia coli. Several also enhanced meropenem-dependent killing of other CPE. Biochemical characterization of a subset indicated the peptides penetrated the bacterial periplasm and directly bound NDM-1 to inhibit enzymatic activity. Additionally, each demonstrated minimal haemolysis and cytotoxicity against mammalian cell lines.
Our approach advances a molecular platform for antimicrobial discovery, which complements the growing need for alternative antimicrobials. We also discovered lead NDM-1 inhibitors, which serve as a starting point for further chemical optimization.
金属β-内酰胺酶（MBLs）是一类新兴的抗菌药物耐药酶，可降解β-内酰胺类抗生素，包括最后一道防线的碳青霉烯类。产碳青霉烯酶肠杆菌科（CPE）引起的感染越来越普遍，但治疗选择有限。虽然几种丝氨酸依赖性β-内酰胺酶抑制剂与常用的β-内酰胺类药物联合使用，但目前尚无 MBL 抑制剂被批准用于联合治疗。因此，迫切需要针对 MBL 以恢复碳青霉烯类药物对 CPE 的活性的新型化合物。在此，我们鉴定并表征了与新型合成肽抑制剂结合并抑制 NDM-1 的新型合成肽抑制剂，NDM-1 是 CPE 中新兴的β-内酰胺类耐药机制。
我们利用表面定位抗菌显示（SLAY）来鉴定和表征抑制 NDM-1 的肽，NDM-1 是 CPE 中主要的碳青霉烯类耐药机制。在存在/不存在碳青霉烯类的情况下，化学合成了先导抑制剂序列，并计算了 MBC 和 MIC。与重组 NDM-1 和选定的肽进行的动力学分析测试了直接结合，并支持了 NDM-1 抑制剂的作用机制。还测试了抑制剂的细胞毒性。
我们鉴定了大约 1700 种序列，这些序列增强了碳青霉烯类对 NDM-1 大肠杆菌的依赖性杀伤作用。其中一些还增强了其他 CPE 对美罗培南的依赖性杀伤作用。对一组亚组的生化特征分析表明，这些肽穿透了细菌的周质，并直接与 NDM-1 结合以抑制酶活性。此外，每种肽都表现出对哺乳动物细胞系的最小溶血和细胞毒性。
我们的方法推进了抗菌药物发现的分子平台，这补充了对抗生素替代品日益增长的需求。我们还发现了 NDM-1 的先导抑制剂，它为进一步的化学优化提供了起点。
