In an attempt to exploit the hydrolytic mechanism by which β-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based... Show moreIn an attempt to exploit the hydrolytic mechanism by which β-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the β-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure–activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity. Show less
Metallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes that inactivate essentially all classes of β-lactam antibiotics including last-resort carbapenems. At present there are no... Show moreMetallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes that inactivate essentially all classes of β-lactam antibiotics including last-resort carbapenems. At present there are no clinically approved MBL inhibitors, and in order to develop such agents it is essential to understand their inhibitory mechanisms. Herein, we describe a comprehensive mechanistic study of a panel of structurally distinct MBL inhibitors reported in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration (IC50) for each inhibitor against MBLs belonging to the NDM and IMP families. In addition, the binding affinities of the inhibitors for Zn2+, Ca2+ and Mg2+ were assessed by using isothermal titration calorimetry (ITC). We also compared the ability of the different inhibitors to resensitize a highly resistant MBLexpressing Escherichia coli strain to meropenem. These investigations reveal clear differences between the MBL inhibitors studied in terms of their IC50 value, metal binding ability, and capacity to synergize with meropenem. Notably, our studies demonstrate that potent MBL inhibition and synergy with meropenem are not explicitly dependent on the capacity of an inhibitor to strongly chelate zinc. Show less
Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we... Show moreFood for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A β-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined. Show less
The rapid emergence of antimicrobial resistance is a major threat to human health. Antibiotics modulate a wide range of biological processes in bacteria and as such, the study of bacterial cellular... Show moreThe rapid emergence of antimicrobial resistance is a major threat to human health. Antibiotics modulate a wide range of biological processes in bacteria and as such, the study of bacterial cellular signaling could aid the development of urgently needed new antibiotic agents. Due to the advances in bacterial phosphoproteomics, such a systemwide analysis of bacterial signaling in response to antibiotics has recently become feasible. Here we present a dynamic view of differential protein phosphorylation upon antibiotic treatment and antibiotic resistance. Most strikingly, differential phosphorylation was observed on highly conserved residues of resistance regulating transcription factors, implying a previously unanticipated role of phosphorylation mediated regulation. Using the comprehensive phosphoproteomics data presented here as a resource, future research can now focus on deciphering the precise signaling mechanisms contributing to resistance, eventually leading to alternative strategies to combat antimicrobial resistance. Show less
Antibiotic resistance caused by β-lactamase production continues to present a growing challenge to the efficacy of β-lactams and their role as the most important class of clinically used... Show moreAntibiotic resistance caused by β-lactamase production continues to present a growing challenge to the efficacy of β-lactams and their role as the most important class of clinically used antibiotics. In response to this threat however, only a handful of β-lactamase inhibitors have been introduced to the market over the past thirty years. The first-generation β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) are all β-lactam derivatives and work primarily by inactivating class A and some class C serine β-lactamases. The newer generations of β-lactamase inhibitors including avibactam and vaborbactam are based on non-β-lactam structures and their spectrum of inhibition is extended to KPC as an important class A carbapenemase. Despite these advances several class D and virtually all important class B β-lactamases are resistant to existing inhibitors. The present review provides an overview of recent FDA-approved β-lactam/β-lactamase inhibitor combinations as well as an update on research efforts aimed at the discovery and development of novel β-lactamase inhibitors. Show less
Metallo-β-lactamases (MBLs) are a growing threat to the continued efficacy of β-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of... Show moreMetallo-β-lactamases (MBLs) are a growing threat to the continued efficacy of β-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of inhibition was not fully characterized. Equilibrium dialysis and metal analysis studies revealed that 2 equiv of AMA effectively removes 1 equiv of Zn(II) from MBLs NDM-1, VIM-2, and IMP-7 when the MBL is at micromolar concentrations. Conversely, 1H NMR studies revealed that 2 equiv of AMA remove 2 equiv of Co(II) from Co(II)-substituted NDM-1, VIM-2, and IMP-7 when the MBL/AMA are at millimolar concentrations. Our findings reveal that AMA inhibits the MBLs by removal of the active site metal ions required for β-lactam hydrolysis among the most clinically significant MBLs. Show less
Hart, P. 't; Wood, T.M.; Tehrani, K.H.M.E.; Harten, R.M. van; Śleszyńska, M.; Rentero Rebollo, I.; ... ; Martin, N.I. 2017
Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a... Show moreCreative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor lipid II. In doing so we identified unique lipid II binding peptides that upon lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides. Show less
