The superbug infection caused by New Delhi metallo-β-lactamase (NDM-1) has grown into an emerging threat, labelling and inhibition of NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report a potent covalent scaffold, ebsulfur, for targeting the protein in vitro and in vivo. Enzymatic kinetic study indicated that eighteen ebsulfurs gained except 1a-b and 1f inhibited NDM-1, exhibiting an IC50 value ranging of 0.16-9 μM, and 1g was found to be the best, dose- and time-dependent inhibitor with an IC50 of 0.16 μM. Also, these ebsulfurs effectively restored the antibacterial activity of cefazolin against E. coli expressing NDM-1, and the best effect was observed to be from 1g, 1i and 1n, resulting in an 256-fold reduction in MIC of the antibiotic at a dose of 16 μg/mL. The equilibrium dialysis study implied that the ebsulfur disrupted the coordination of one Zn(II) ion at active site of NDM-1. Labelling of NDM-1 using a constructed fluorescent ebsulfur Ebs-R suggested that the inhibitor covalently bound to the target through SDS-PAGE analysis in vitro. Also, labelling NDM-1 in living E. coli cells with Ebs-R by confocal microscopic imaging showed the real-time distribution change process of intracellular recombinant protein NDM-1. Moreover, the cytotoxicity of these ebsulfurs against L929 mouse fibroblastic cells was tested, and their capability to restore antibacterial activity of antibiotic against clinical strains E. coli EC08 producing NDM-1 was determined. The ebsulfur scaffold proposed here is valuable for development of the covalent irreversible inhibitors of NDM-1, and also for labelling the target in vitro and in vivo.

中文翻译：

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作为体外和体内抑制和标记新德里金属β-内酰胺酶-1的有效支架，硫是有效的。

由新德里金属β-内酰胺酶（NDM-1）引起的超级细菌感染已发展成为一种新兴威胁，由于其在病原细菌之间的穿梭作用，对NDM-1的标记和抑制已证明具有挑战性。在这里，我们报道了一种有效的共价支架ebsulfur，可用于在体内和体外靶向蛋白质。酶动力学研究表明，除了1a-b和1f抑制NDM-1之外，还获得了18种依硫磺，其IC50值为0.16-9μM，并且发现1g是最佳的剂量和时间依赖性抑制剂，IC50为0.16微米 而且，这些依硫磺有效地恢复了头孢唑啉对表达NDM-1的大肠杆菌的抗菌活性，并且观察到的最佳效果是从1g，1i和1n起，导致抗生素的MIC剂量降低256倍。 16微克/毫升。平衡透析研究表明，乙硫醚破坏了NDM-1活性位点上一个Zn（II）离子的配位。使用构建的荧光乙硫Ebs-R标记NDM-1，表明该抑制剂通过体外SDS-PAGE分析共价结合至靶标。此外，通过共聚焦显微镜成像在活的大肠杆菌细胞中用Ebs-R标记NDM-1，显示了细胞内重组蛋白NDM-1的实时分布变化过程。此外，测试了这些依硫磺对L929小鼠成纤维细胞的细胞毒性，并确定了它们恢复抗生素对临床菌株E. coli EC08产生NDM-1的抗菌活性的能力。本文提出的乙硫支架对于开发NDM-1的共价不可逆抑制剂具有重要意义，