Development of Class IIa Bacteriocins as Therapeutic Agents

Class IIa bacteriocins have been primarily explored as natural food preservatives, but there is much interest in exploring the application of these peptides as therapeutic antimicrobial agents. Bacteriocins of this class possess antimicrobial activity against several important human pathogens. Therefore, the therapeutic development of these bacteriocins will be reviewed. Biological and chemical modifications to both stabilize and increase the potency of bacteriocins are discussed, as well as the optimization of their production and purification. The suitability of bacteriocins as pharmaceuticals is explored through determinations of cytotoxicity, effects on the natural microbiota, and in vivo efficacy in mouse models. Recent results suggest that class IIa bacteriocins show promise as a class of therapeutic agents

Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase–catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in ß-lactam biosynthesis

2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of β-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of β-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of β-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in β-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in β-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in ß-lactam biosynthesis

2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of β-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of β-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of β-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in β-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in β-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms

A Fluorescence‐based assay for screening β‐lactams targeting the Mycobacterium tuberculosis transpeptidase LdtMt2

Mycobacterium tuberculosis L,D‐transpeptidases (Ldts), which are involved in cell wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence‐based assay for LdtMt2, which is suitable for high‐throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2, making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of β‐lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β‐lactams are more potent Ldt inhibitors than other β‐lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure‐activity relationship studies on Ldts, facilitating the identification of new antibiotics active against M. tuberculosis

A Fluorescence‐based assay for screening β‐lactams targeting the Mycobacterium tuberculosis transpeptidase LdtMt2

Mycobacterium tuberculosis L,D‐transpeptidases (Ldts), which are involved in cell wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence‐based assay for LdtMt2, which is suitable for high‐throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2, making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of β‐lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of β‐lactams are more potent Ldt inhibitors than other β‐lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure‐activity relationship studies on Ldts, facilitating the identification of new antibiotics active against M. tuberculosis

Mechanistic insights into β-lactamase-catalysed carbapenem degradation through product characterisation

β-Lactamases are a major threat to the clinical use of carbapenems, which are often antibiotics of last resort. Despite this, the reaction outcomes and mechanisms by which β-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a β-lactam ring fused to a pyrroline ring. Following β-lactamase-mediated opening of the β-lactam, the pyrroline may interconvert between an enamine (2-pyrroline) form and two epimeric imine (1-pyrroline) forms; previous crystallographic and spectroscopic studies have reported all three of these forms in the contexts of hydrolysis by different β-lactamases. As we show by NMR spectroscopy, the serine β-lactamases (KPC-2, SFC-1, CMY-10, OXA-23, and OXA-48) and metallo-β-lactamases (NDM-1, VIM-1, BcII, CphA, and L1) tested all degrade carbapenems to preferentially give the Δ² (enamine) and/or (R)-Δ¹ (imine) products. Rapid non-enzymatic tautomerisation of the Δ² product to the (R)-Δ¹ product prevents assignment of the nascent enzymatic product by NMR. The observed stereoselectivity implies that carbapenemases control the form of their pyrroline ring intermediate(s)/product(s), thereby preventing pyrroline tautomerisation from inhibiting catalysis