产生金属-β-内酰胺酶(MBL)的肠杆菌科是严重的临床关注,特别是没有被批准用于临床的金属-β-内酰胺酶抑制剂。因此,发现和开发MBL抑制剂以恢复可用β-内酰胺的功效是必要的。我们研究了锌螯合部分,1,4,7-三氮杂环壬烷(TACN),对临床耐碳青霉烯类肠杆菌的抑制活性,最低杀菌浓度(MBC),血清效应,分数抑制浓度指数,根据临床和实验室标准研究所(CSLI)指南,使用肉汤微量稀释技术确定时间杀死动力学。使用分光光度法测定TACN的酶动力学参数和细胞毒性作用。通过计算研究了酶-TACN复合物的相互作用。美罗培南恢复了对产生碳青霉烯酶的肠杆菌科细菌的活性,在TACN存在下,MIC从8至64mg/L降低至0.03mg/L。TACN-美罗培南组合显示出MBC/MIC比值≤4的杀菌作用,并观察到协同活性。人类血清对中等收入国家的影响微不足道,发现TACN在高于MIC值的浓度下无细胞毒性。计算研究预测TACN通过靶向其催化活性位点口袋来抑制MBL。这得到了其抑制常数(Ki)的支持,即0.044μM,及其失活常数(Kinact),为0.0406min-1,表明TACN有效抑制MBL,并有望成为潜在的抑制剂。重要性耐碳青霉烯类肠杆菌(CRE)介导的感染仍然是一个重要的公共卫生问题,据报道在世界卫生组织的研究和开发新抗生素的优先病原体清单中至关重要。CRE产生酶,如金属-β-内酰胺酶(MBL),使β-内酰胺抗生素失活。涉及β-内酰胺抗生素和β-内酰胺酶抑制剂的组合疗法仍然是由产生β-内酰胺酶的生物体引起的感染的主要治疗选择。目前,对于MBL阳性细菌感染,目前尚无MBL抑制剂-β-内酰胺联合治疗。因此,开发能够抑制这些酶的有效分子可能是克服这种现象的有希望的方法。TACN通过增强碳青霉烯的活性在测试分子对CREs的抑制活性中起重要作用。这项研究表明,TACN可以有效抑制MBL,并有望成为潜在的MBL抑制剂,以帮助遏制由MBL产生的CRE带来的全球健康威胁。
Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (
TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of
TACN were determined using spectrophotometric assays. The interactions of the enzyme-
TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The
TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that
TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization\'s priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.