Potent Β-lactamase Inhibitor Research Articles

Frameshift Mutations in Genes Encoding PBP3 and PBP4 Trigger an Unusual, Extreme β-Lactam Resistance Phenotype in Burkholderia multivorans.

In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To explore the molecular mechanisms leading to this phenotype, we performed whole genome sequencing (WGS) and microbiological and biochemical assays. WGS analysis revealed that strain AU28442 produced two β-lactamases, AmpC22 and a novel PenA-like β-lactamase denominated PenA39. Additionally, the strain presented frame-shift mutations in the genes encoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic susceptibilities of the parent AU28442 strain carrying blaPenA39 vs the isogenic E. colistrain producing blaPenA39 were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with kcat values of ≤8.8 s-1. An overlay of the crystal structure of PenA39 with PenA1 revealed a shift in the SDN loop in the variant, which may affect the catalytic efficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic examination of AU28442 revealed shortened rod-shaped cells compared to B. multivoransATCC 17616, which carries a full complement of intact PBPs. Further complementation assays confirmed that the loss of PBP3 and PBP4 was the main factor contributing to the high-level β-lactam resistance observed in B. multivoransAU28442. This information allowed us to revert susceptibility by pairing a potent β-lactamase inhibitor with a β-lactam with promiscuous PBP binding. This detailed characterization of B. multivoransprovides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it.

Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus

Clavulanic acid is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus, widely used in combination with β-lactam antibiotics to combat antimicrobial resistance. This systematic review analyzes the most successful methodologies for clavulanic acid overproduction, focusing on the highest yields reported in bench-scale and bioreactor-scale fermentations. Studies have demonstrated that glycerol is the preferred carbon source for clavulanic acid production over other sources like starch and dextrins. The optimization of feeding strategies, especially in fed-batch operations, has improved glycerol utilization and extended the clavulanic acid production phase. Organic nitrogen sources, particularly soybean protein isolates and amino acid supplements such as L-arginine, L-threonine, and L-glutamate, have been proven effective at increasing CA yields both in batch and fed-batch cultures, especially when balanced with appropriate carbon sources. Strain engineering approaches, including mutagenesis and targeted genetic modifications, have allowed for the obtainment of overproducer S. clavuligerus strains. Specifically, engineering efforts that overexpress key regulatory genes such as ccaR and claR, or that disrupt competing pathways, redirect the metabolic flux towards CA biosynthesis, leading to high clavulanic acid titers. The fed-batch operation at the bioreactor scale emerges as the most feasible alternative for prolonged clavulanic acid production with both wild-type and mutant strains, allowing for the attainment of high titers during cultivations.

Chemically synthesised flavone and coumarin based isoxazole derivatives as broad spectrum inhibitors of serine β-lactamases and metallo-β-lactamases: a computational, biophysical and biochemical study

The β-lactam antibiotics are the most effective medicines for treating bacterial infections. Resistance to them, particularly through the production of β-lactamases, which can hydrolyse all kinds of β-lactams, poses a threat to their continued use. The synthesised flavone and coumarin based isoxazole derivatives have the potential to be used as broad-spectrum inhibitors of the mechanistically different serine-(SBL) and metallo-β-lactamases (MBL). The synthesised compounds were discovered as potent β-lactamase inhibitors using molecular docking and in silico pharmacokinetic analysis. We studied the binding of chemically synthesised inhibitors to clinically significant β-lactamases of class A, B, and C using biophysical and biochemical approaches, and computational analyses. These molecules follow Lipinski’s rule of five and have acceptable solubility, permeability, and oral bioavailability. These molecules were found to be non-toxic and non-carcinogenic. MIC results suggest that these molecules restore the antibiotic efficacy against class A, B, and C β-lactamases. Kinetics data showed that these molecules reduce the catalytic efficiency of clinically relevant class A, B, and C β-lactamases. Fluorescence study showed significant interaction between these flavone-/coumarin-based isoxazole derivatives and class A/B/ C β-lactamases. This study showed promising effect of these new generation compounds as broad spectrum β-lactamase inhibitors of both SBLs and MBLs. Communicated by Ramaswamy H. Sarma

The role of new carbapenem combinations in the treatment of multidrug-resistant Gram-negative infections.

Multi-drug resistant (MDR) Gram-negative bacteria represent a growing threat, with an increasing prevalence of carbapenem-resistant Enterobacterales (CRE) infections, for which treatment options are limited. New treatment combinations composed of a β-lactam antibiotic plus a potent β-lactamase inhibitor (BLI) with anti-carbapenemase activity have been developed, including two carbapenem/BLI combinations that are commercially available—meropenem/vaborbactam (Vabomere® in the US, Vaborem® in Europe; Melinta Therapeutics) and imipenem/cilastatin/relebactam (Recarbrio®; Merck Sharp & Dohme), plus one other (meropenem/nacubactam) in early clinical development. This review provides a summary of the preclinical evidence supporting the use of carbapenem/BLI combinations and presents the clinical evidence across a range of MDR Gram-negative infections, with a focus on the use of meropenem/vaborbactam. All three BLIs have shown in vivo activity against Klebsiella pneumoniae carbapenemase and other class A carbapenemases. In 2019, meropenem/vaborbactam was listed in the WHO’s list of essential medicines, because of its activity against priority 1 antibiotic-resistant pathogens. Meropenem/vaborbactam has considerable in vitro and in vivo activity against CRE, and in vitro evidence showing a low potential for resistance at clinically relevant doses. In randomized trials, meropenem/vaborbactam was non-inferior to piperacillin/tazobactam in patients with complicated urinary tract infection and more effective than the best-available treatment in patients with serious CRE infections. Meropenem/vaborbactam is well tolerated and, based on clinical experience, demonstrated lower toxicity compared with the combination regimens that have previously been the standard of care. In conclusion, carbapenem/BLI combinations represent an important therapeutic strategy in patients with MDR Gram-negative infections.

Structural analysis of the boronic acid β-lactamase inhibitor vaborbactam binding to Pseudomonas aeruginosa penicillin-binding protein 3.

Antimicrobial resistance (AMR) mediated by β-lactamases is the major and leading cause of resistance to penicillins and cephalosporins among Gram-negative bacteria. β-Lactamases, periplasmic enzymes that are widely distributed in the bacterial world, protect penicillin-binding proteins (PBPs), the major cell wall synthesizing enzymes, from inactivation by β-lactam antibiotics. Developing novel PBP inhibitors with a non-β-lactam scaffold could potentially evade this resistance mechanism. Based on the structural similarities between the evolutionary related serine β-lactamases and PBPs, we investigated whether the potent β-lactamase inhibitor, vaborbactam, could also form an acyl-enzyme complex with Pseudomonas aeruginosa PBP3. We found that this cyclic boronate, vaborbactam, inhibited PBP3 (IC50 of 262 μM), and its binding to PBP3 increased the protein thermal stability by about 2°C. Crystallographic analysis of the PBP3:vaborbactam complex reveals that vaborbactam forms a covalent bond with the catalytic S294. The amide moiety of vaborbactam hydrogen bonds with N351 and the backbone oxygen of T487. The carboxyl group of vaborbactam hydrogen bonds with T487, S485, and S349. The thiophene ring and cyclic boronate ring of vaborbactam form hydrophobic interactions, including with V333 and Y503. The active site of the vaborbactam-bound PBP3 harbors the often observed ligand-induced formation of the aromatic wall and hydrophobic bridge, yet the residues involved in this wall and bridge display much higher temperature factors compared to PBP3 structures bound to high-affinity β-lactams. These insights could form the basis for developing more potent novel cyclic boronate-based PBP inhibitors to inhibit these targets and overcome β-lactamases-mediated resistance mechanisms.

Development of a Practical Manufacturing Process to Relebactam via Thorough Understanding of the Origin and Control of Oligomeric Impurities

The development of a robust manufacturing process to relebactam, a potent β-lactamase inhibitor, is described. The origin and control strategy of oligomer impurities formed during hydrogenolysis of benzyl urea intermediate 9 and deprotection of Boc-sulfate intermediate 11 have been thoroughly studied and developed. The optimal manufacturing process has been successfully implemented for commercialization, preparing relebactam (1) in >99% purity with a non-detectable level of oligomers.

Design and enantioselective synthesis of 3-(α-acrylic acid) benzoxaboroles to combat carbapenemase resistance.

Chiral 3-substituted benzoxaboroles were designed as carbapenemase inhibitors and efficiently synthesised via asymmetric Morita–Baylis–Hillman reaction. Some of the benzoxaboroles were potent inhibitors of clinically relevant carbapenemases and restored the activity of meropenem in bacteria harbouring these enzymes. Crystallographic analyses validate the proposed mechanism of binding to carbapenemases, i.e. in a manner relating to their antibiotic substrates. The results illustrate how combining a structure-based design approach with asymmetric catalysis can efficiently lead to potent β-lactamase inhibitors and provide a starting point to develop drugs combatting carbapenemases.

Activity of Meropenem with a Novel Broader-Spectrum β-Lactamase Inhibitor, WCK 4234, against Gram-Negative Pathogens Endemic to New York City.

WCK 4234 is a novel diazabicyclooctane with potent inhibitory activity against class A and D carbapenemases and class C enzymes. We examined the in vitro activity of meropenem plus WCK 4234 (4 or 8 μg/ml) against Gram-negative pathogens from New York City. Three groups of isolates were analyzed: a contemporary collection of isolates, a collection of known carbapenem-resistant isolates, and a collection of isolates with defined resistance mechanisms. From the contemporary collection, we found (i) that all Enterobacteriaceae were susceptible to meropenem plus WCK 4234, (ii) that susceptibility rates for Acinetobacter baumannii were 56.5% for meropenem alone, 82.6% with 4 μg/ml WCK 4234, and 95.7% with 8 μg/ml WCK 4234, and (iii) that WCK 4234 had a modest effect on susceptibility of Pseudomonas aeruginosa Against a collection of carbapenem-resistant isolates, the addition of WCK 4234 to meropenem (i) restored meropenem susceptibility against Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates, (ii) improved susceptibility against A. baumannii, and (iii) had a negligible effect against P. aeruginosa When tested against isolates with defined mechanisms of resistance, MICs of meropenem plus WCK 4234 were higher for K. pneumoniae with blaKPC albeit well below the susceptibility breakpoint; efflux systems or porins did not correlate with susceptibility. For A. baumannii, MICs of meropenem plus WCK 4234 did not correlate with efflux systems, outer membrane protein, blaampC, or blaoxa-51; however, MICs were higher in isolates with extended-spectrum β-lactamases (ESBLs). For P. aeruginosa, isolates with relatively higher MICs of meropenem plus WCK 4234 had increased expression of ampC WCK 4234 is a potent β-lactamase inhibitor that, when combined with meropenem, displays promising activity against multidrug-resistant pathogens.

Diastereoselective FeCl3·6H2O/NaBH4 Reduction of Oxime Ether for the Synthesis of β-Lactamase Inhibitor Relebactam.

Relebactam, a potent β-lactamase inhibitor, in combination with Primaxin is an FDA-approved (Recarbrio) treatment for serious and antibiotic-resistant bacterial infections. An efficient synthesis of key chiral piperidine intermediate 1 suitable for large-scale preparation of relebactam is described. The key steps include a unique highly diastereoselective FeCl3·6H2O/NaBH4 reduction of a chiral oxime ether and chemoselective amidation of the resulting unprotected pipecolic acid. Nuclear magnetic resonance studies and density functional theory calculations were carried out on the substrate-Fe(III) complexes, which shed light on diastereoselective reduction.

Improved Preparation of a Key Hydroxylamine Intermediate for Relebactam: Rate Enhancement of Benzyl Ether Hydrogenolysis with DABCO

Previous methods to prepare a bicyclic N-hydroxyl urea intermediate in the synthesis of the potent β-lactamase inhibitor relebactam were effective, but deemed unsuitable for long-term use. Therefore, we developed an in situ protection protocol during hydrogenolysis and a robust deprotection/isolation sequence of this unstable intermediate employing a reactive crystallization. During the hydrogenation studies, we discovered a significant rate enhancement of O-benzyl ether hydrogenolysis in the presence of organic amine bases, especially DABCO. The broader utility of the application of organic bases on the hydrogenolysis of a range of O- and N-benzyl-containing substrates was demonstrated.

Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering.

ABSTRACTThe emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” β-lactams, preferentially binding β-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel β-lactams and β-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases.

The Novel β-Lactamase Inhibitor, ETX-2514, in Combination with Sulbactam Effectively Inhibits Acinetobacter baumannii

BackgroundMultidrug resistant (MDR) Acinetobacter sp. were deemed a “serious” health threat by the Centers for Disease Control and Prevention with a daunting 63% of infections being nearly untreatable. Underlying this challenging pathogen are the presence of a chromosomal class C β-lactamase, Acinetobacter-derived cephalosporinase (ADC), as well as the abundant prevalence of class D OXA β-lactamases that hydrolyze carbapenems in conjunction with a lack of potent β-lactamase inhibitors. Based on the ability of ETX2514, a rationally designed novel diazabicyclooctane inhibitor (Figure 1A), to inhibit class D β-lactamases as well as PBPs, we hypothesized that highly resistant clinical isolates of A. baumannii will demonstrate susceptibility to the sulbactam-ETX2514 combination and that A. baumannii β-lactamases, ADC-7 and OXA-58 will be readily inactivated by ETX2514.MethodsSusceptibility testing according to Clinical and Laboratory Standards Institute was performed for sulbactam ±4 mg/L ETX2514 using 72 A. baumannii strains. More than half of the isolates are MDR, have ≥ eight resistant determinants, contain an ADC β-lactamase, and have OXA-23 and OXA-58-like β-lactamases in the carbapenem resistant isolates. ADC-7 and OXA-58 β-lactamases were purified and characterized with ETX2514 by steady-state inhibition kinetics and Q-TOF mass spectrometry.ResultsThe A. baumannii strains demonstrated an MIC90 of 32 mg/L for sulbactam and 2 mg/L for the sulbactam-ETX-2514 combination. The addition of ETX2514 lowered the MIC50 from 8 to 1 mg/L (Figure 1B). ETX-2514 effectively inhibited purified OXA-58 (k2/K = 2.5 ± 0.3 × 105 M−1 s−1 and Ki apP = 0.39 ± 0.01 µM) and ADC-7 (k2/K =1.0 ± 0.1 × 106 M−1 s−1 and Ki app = 0.11 ± 0.04 µM). The two β-lactamases displayed similar dissociation constants (Kd =1 ± 0.1 nM), but ADC-7 possessed a faster dissociation rate (koff =8 ± 1 × 10−4 s−1 for ADC-7 and 1.6 ± 0.3 × 10−4 s−1for OXA-58). Using mass spectrometry, ADC-7 and OXA-58 were found to stably bind the ETX-2514 compound for up to 24 hours (Figure 1C).ConclusionETX2514 is a new β-lactamase inhibitor that is strikingly effective at restoring susceptibility to highly drug-resistant A. baumannii isolates when combined with sulbactam via inhibition of the ADC-7 and OXA-58 β-lactamases.Disclosures F. Van Den Akker, Entasis: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant; K. M. Papp-Wallace, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant; R. A. Bonomo, Entasis: Grant Investigator, Research grant Allecra: Grant Investigator, Research grant Wockhardt: Grant Investigator, Research grant Merck: Grant Investigator, Research grant Roche: Grant Investigator, Research grant GSK: Grant Investigator, Research grant Allergan: Grant Investigator, Research grant Shionogi: Grant Investigator, Research grant

Design, synthesis and characterization of peptidyl boronate analogues as effective antimicrobial agents

Antibiotic-resistant penicillin binding protein (PBPs) production is one of the reasons why bacteria develop resistance to β-lactam antibiotics, and this needs to be tackled in the continual battle to produce effective antibiotics. The transition state analogue of boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates are known to be potent β-lactamase inhibitors. We have recently identified boronic acids as a selective scaffold for Actinomadura sp. strain R39 DD-peptidase (PBP). Here, we report the synthesis and biological evaluation of a comprehensive set of 16 boronic acid analogues and establish their structure–activity relationships as well as their potential for use as PBP inhibitors. The docking studies of all the synthesized compounds were carried out in Molegro Virtual Docker. Of the 16 compounds synthesized, compound 8e exhibits the highest binding affinity (187.5 kcal/mol) followed by compounds 8f, 8d, 8a, 8g, 8c and 8i. The minimum inhibitory concentration (MIC) of all the 16 synthesised compounds 6a–6f, 8a–8i, 10 and 11 were tested against two Gram-positive (S. aureus, and S. pyogene) and eight Gram-negative (E. coli, C. koseri, K. oxytoca, K. pneumoniae, S. dysentriae, S. typhi, C. fruendi, and P. aeruginosa) bacteria. The biological evaluation of the synthesized boronic acid analogues were found to be effective in all cases, although it is more pronounced for pyrazine-containing dipeptidyl boronic acid (8e, MIC = 0.062 mg/ml) and hence considered as an effective inhibitor, thereby highlighting the importance of phenylalanine as linker and heterocyclic aromatic carboxylic acid as terminal moieties. This study exemplifies that pyrazine-containing dipeptidyl boronic acid (8e) as a PBP inhibitor could open up new avenues in the development of PBP-inhibiting molecules that eventually display bactericidal effects on distinct bacterial species.

Real-time activity assays of β-lactamases in living bacterial cells: application to the inhibition of antibiotic-resistant E. coli strains.

The emergence of antibiotic resistance caused by β-lactamases, including serine β-lactamases (SβLs) and metallo-β-lactamases (MβLs), is a global public health threat. L1, a B3 subclass MβL, hydrolyzes almost all of known β-lactam antibiotics. We report a simple and straightforward UV-Vis approach for real-time activity assays of β-lactamases inside living bacterial cells, and this method has been exemplified by choosing antibiotics, L1 enzyme, Escherichia coli expressing L1 (L1 E. coli), Escherichia coli expressing extended-spectrum β-lactamases (ESBL-E. coli), clinical bacterial strains, and reported MβL and SβL inhibitors. The cell-based studies demonstrated that cefazolin was hydrolyzed by L1 E. coli and clinical strains, and confirmed the hydrolysis to be inhibited by two known L1 inhibitors EDTA and azolylthioacetamide (ATAA), with an IC50 value of 1.6 and 18.9 μM, respectively. Also, it has been confirmed that the breakdown of cefazolin caused by ESBL-E. coli was inhibited by clavulanic acid, the first SβL inhibitor approved by FDA. The data gained through this approach are closely related to the biological function of the target enzyme in its physiological environment. The UV-Vis method proposed here can be applied to target-based whole-cell screening to search for potent β-lactamase inhibitors, and to assays of reactions in complex biological systems, for instance in medical assays.

Clavulanic Acid Production by Streptomyces clavuligerus using Solid State Fermentation on Polyurethane Foam

Clavulanic acid (CA), a metabolite of Streptomyces clavuligerus , is a potent β-lactamase inhibitor. In this study, polyurethane foam (PUF) was used as inert solid support to produce clavulanic acid by solid state fermentation (SSF). Maximal CA yield of 263 µg/ml was obtained at pH 6.5, incubation temperature 29°C, 10 ml medium per 3 g PUF, 0.015% added glycerol, 2% added lithium chloride (LiCl), and 2 g/L added ornithine. Under the same conditions, the yield of CA produced by SSF on PUF is apparently higher than that by submerged fermentation (SMF). In addition, CA produced by using this method is of higher purity and easier to be extracted. Citation: Wang, H. and Chen, H. (2016). Clavulanic Acid Production by Streptomyces clavuligerus using Solid State Fermentation on Polyurethane Foam. Trends in Renewable Energy, 2(1), 2-12. DOI: 10.17737/tre.2016.2.1.0018

Investigation of the in vivo interaction between β-lactamaseand its inhibitor protein

The affinity of β-lactamase inhibitory protein (BLIP) for TEM-1 β-lactamase has raised hopes in the challenge of protein- based inhibitor discovery for β-lactamase-mediated antibiotic resistance. Currently, the effect of the formation of the β-lactamase:BLIP complex in vivo in β-lactam resistant bacteria is an open question. The scarcity of information to the extent to which BLIP can impair β-lactamase activity inside cells has urged us to assess the in vivo efficacy of BLIP as a potent β-lactamase inhibitor. To this end, β-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of β-lactamase and BLIP and the formation of the TEM-1 β-lactamase:BLIP complex in the periplasmic space of E. coli were verified by electrophoretic and Western blot techniques. Growth profiles of the cells expressing both β-lactamase and its protein inhibitor, complemented with β-lactamase activity measurements, suggested that BLIP synthesis retarded cell growth and reduced β-lactamase activity. Although co-expression of β-lactamase and its protein inhibitor did not completely impair cell growth, the specificity of BLIP enabled it to bind β-lactamase in the bacterial periplasm, regardless of the crowding components.

Clavulanic acid production by Streptomyces clavuligerus: biogenesis, regulation and strain improvement

Clavulanic acid (CA) is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus and has been successfully used in combination with β-lactam antibiotics (for example, Augmentin) to treat infections caused by β-lactamase-producing pathogens. Since the discovery of CA in the late 1970s, significant information has accumulated on its biosynthesis, and regarding molecular mechanisms involved in the regulation of its production. Notably, the genes directing CA biosynthesis are clustered along with the genes responsible for the biosynthesis of the β-lactam antibiotic, cephamycin C, and co-regulated, which makes this organism unique in that the production of an antibiotic and production of a small molecule to protect the antibiotic from its enzymatic degradation are controlled by shared mechanisms. Traditionally, the industrial strain improvement programs have relied significantly on random mutagenesis and selection approach. However, the recent availability of the genome sequence of S. clavuligerus along with the capability to build metabolic models, and ability to engineer the organism by directed approaches, has created exciting opportunities to improve strain productivity more efficiently. This review will include focus mainly on the gene organization of the CA biosynthetic genes, regulatory mechanisms that affect its production, and will include perspectives on improving strain productivity.

Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C

In response to the widespread use of β-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent β-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC50: 1.3μM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC50=26μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC50=138μM) and a class C PBP (R39 from Actinomadura sp., IC50=0.6μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.

Rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces clavuligerus

Clavulanic acid is a potent β-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of l-arginine and d-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces clavuligerus. Two genes ( gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.

In vitro activities of novel oxapenems, alone and in combination with ceftazidime, against gram-positive and gram-negative organisms.

Four novel oxapenem compounds (i.e., AM-112, AM-113, AM-114, and AM-115) were investigated for their beta-lactamase inhibitory activity against a panel of isolated class A, C, and D enzymes, which included expanded-spectrum beta-lactamase enzymes (ESBLs). The oxapenems were potent beta-lactamase inhibitors. Activity varied within the group, with AM-113 and AM-114 proving to be the most active compounds. The 50% inhibitory concentrations for these agents were up to 100,000-fold lower than that of clavulanic acid against class C and D enzymes. As a group, the oxapenems were more potent than clavulanic acid against enzymes from all classes. The ability of these compounds to protect ceftazidime from hydrolysis by beta-lactamase-producing strains was evaluated by MIC tests that combined ceftazidime and each oxapenem in a 1:1 or 2:1 ratio. The oxapenems markedly reduced the MICs for ceftazidime against class C hyperproducing strains and strains producing TEM- and SHV-derived ESBLs. There was little difference between the activity of 1:1 and 2:1 combinations of ceftazidime and oxapenem. The oxapenems failed to enhance the activity of ceftazidime against derepressed AmpC-producing Pseudomonas aeruginosa strains.