Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-β-lactamase‑1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems

Carbapenem
resistance mediated by metallo-β-lactamases (MBL)
such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a
major factor threatening the efficacy of essential β-lactam
antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy-
and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors
of NDM-1 with highly improved inhibitory activity and binding affinity.
The most active compounds formed reversibly inactive ternary protein-inhibitor
complexes with two zinc ions as proven by native protein mass spectrometry
and bio-layer interferometry. Modification of the NDM-1 structure
with remarkable entropic gain was shown by isothermal titration calorimetry
and NMR spectroscopy of isotopically labeled protein. The best compounds
were potent inhibitors of NDM-1 and other representative MBL with
no or little inhibition of human zinc-binding enzymes. These inhibitors
significantly reduced the minimum inhibitory concentrations (MIC)
of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM‑1 as well as for several multidrug-resistant
clinical strains at concentrations non-toxic to human cells.

由金属β-内酰胺酶（metallo-β-lactamases, MBL）——如新德里金属β-内酰胺酶-1（New Delhi metallo-β-lactamase-1, NDM-1）——介导的碳青霉烯类抗生素耐药性，已成为威胁核心β-内酰胺类抗生素临床疗效的关键因素。本研究以先导片段吡啶二羧酸（dipicolinic acid, DPA）为起点，开发得到8-羟基喹啉-2-羧酸与8-磺酰胺基喹啉-2-羧酸类NDM-1抑制剂，其抑制活性与结合亲和力均得到大幅提升。经天然蛋白质谱法与生物层干涉术验证，活性最优的化合物可与两个锌离子结合，形成可逆的失活三元蛋白-抑制剂复合物。通过等温滴定量热法与同位素标记蛋白核磁共振波谱法分析，证实此类化合物可使NDM-1的结构发生显著熵增益修饰。最优化合物对NDM-1及其他代表性金属β-内酰胺酶均展现出强效抑制活性，且对人源锌结合酶几乎无抑制作用或抑制效果极弱。在对人体细胞无毒的给药浓度下，此类抑制剂可显著降低美罗培南对重组表达blaNDM-1的多重耐药菌，以及多株临床分离多重耐药菌的最低抑菌浓度（minimum inhibitory concentrations, MIC）。