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The effective strategies to neutralize the New Delhi metallo-β-lactamase (NDM-1) activity offer unique opportunities to combination therapy because NDM-1 inactivates all classes of carbapenem antibiotics, which are widely regarded as the last resort of drugs for treating serious bacterial infections. Here we describe the efficient construction of a series of trans-1,3,4-trisubstituted pyrrolidines via boric acid-catalyzed 1,3-dipolar cycloaddition of N-benzylazomethine ylide with methyl ferulate for the biological evaluation of their cytotoxicity and synergistic activity in combination with meropenem towards NDM-1 positive carbapenem-resistant Enterobacteriaceae (CRE). The cell-based screens generated one promising hit, namely compound 10e, which exhibited low cytotoxicity (IC50 > 128 μM), moderate NDM-1 enzyme inhibition (IC50 = 51 μM), and potent synergistic activity against a panel of clinically isolated NDM-1 positive CRE with fractional inhibitory concentration indexes ranging from 0.01 to 0.25. Structure–activity relationship studies revealed that the zinc-chelating moiety of 2-(bis(pyridin-2-ylmethyl)-amino)acetyl group of compound 10e plays a pivotal role for potent activity. Regarding the inhibition mechanism, a series of biochemical assays revealed that compound 10e may inactivate NDM-1 activity by displacing both zinc ions from the active site of the enzyme. Altogether, our studies indicate that compound 10e represents an important pyrrolidine-type scaffold targeting NDM-1, providing a promising starting point to be further developed as carbapenem antibiotic adjuvants. [ABSTRACT FROM AUTHOR]

Antibiotic resistance mediated by β-lactamases including metallo-β-lactamases (MβLs) has become an emerging threat. In this study, we employed UV-vis to monitor the activity and inhibition of New Delhi MβL (NDM), K. pneumoniae carbapenemase (KPC) and MβL VIM-2 in live bacterial cells in real time. The monitoring showed that the faropenem was hydrolyzed by six strains which express β-lactamases, K. pneumoniae, P. aeruginosa, E. coli expressing extended-spectrum β-lactamase (ESBL), NDM-1 (EC07), VIM-2, and methicillin-resistant S. aureus (MRSA), but not by E. coli and S. aureus which do not express β-lactamases. Also, four β-lactams cefazolin, meropenem, faropenem and amoxicillin were hydrolyzed by the NDM and KPC expressed in K. pneumoniae. Cell-based studies revealed that the targets NDM and KPC expressed in K. pneumoniae cells were inhibited by three known inhibitors ethylene diamine tetraacetic acid (EDTA), ebselen and clavulanic acid, with a fifty percent inhibitory concentration (IC50) of 1.2, 84.7 and 13.3 μM, and the VIM-2 expressed in E. coli cells was inhibited by EDTA, D -captopril and rhodanine with an IC50 of 1.3, 510.8 and 9.3 μM, respectively. This study provides an approach that could be applied to screen and evaluate small molecule inhibitors of β-lactamases in whole cells or to identify drug resistant bacteria. [ABSTRACT FROM AUTHOR]