New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC50 values against DNA gyrase, and submicromolar IC50 values against topoisomerase IV from Escherichia coil and Staphylococcus aureus. The most potent compound in the series has an IC50 value of 13 nM against E. coil gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative with an IC50 value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 mu M against Enterococcus faecalis, and 3.13 mu M against wild type S. aureus, methicillinresistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PA beta N) is 4.6 mu M. (C) 2018 Elsevier Masson SAS. All rights reserved

New N-phenylpyrrolamide DNA gyrase B inhibitors: Optimization of efficacy and antibacterial activity

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC50 values against DNA gyrase, and submicromolar IC50 values against topoisomerase IV from Escherichia coil and Staphylococcus aureus. The most potent compound in the series has an IC50 value of 13 nM against E. coil gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative with an IC50 value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 mu M against Enterococcus faecalis, and 3.13 mu M against wild type S. aureus, methicillinresistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PA beta N) is 4.6 mu M. (C) 2018 Elsevier Masson SAS. All rights reserved.Peer reviewe

An optimised series of substituted N-phenylpyrrolamides as DNA gyrase B inhibitors

ATP competitive inhibitors of DNA gyrase and topoisomerase IV have great therapeutic potential, but none of the described synthetic compounds has so far reached the market. To optimise the activities and physicochemical properties of our previously reported N-phenylpyrrolamide inhibitors, we have synthesized an improved, chemically variegated selection of compounds and evaluated them against DNA gyrase and topoisomerase IV enzymes, and against selected Gram-positive and Gram-negative bacteria. The most potent compound displayed IC50 values of 6.9 nM against Escherichia coli DNA gyrase and 960 nM against Staphylococcus aureus topoisomerase IV. Several compounds displayed minimum inhibitory concentrations (MICs) against Gram-positive strains in the 1-50 mu M range, one of which inhibited the growth of Enterococcus faecalis, Enterococcus faecium, S. aureus and Streptococcus pyogenes with MIC values of 1.56 mu M, 1.56 mu M, 0.78 mu M and 0.72 mu M, respectively. This compound has been investigated further on methicillin-resistant S. aureus (MRSA) and on ciprofloxacin non-susceptible and extremely drug resistant strain of S. aureus (MRSA VISA). It exhibited the MIC value of 2.5 mu M on both strains, and MIC value of 32 mu M against MRSA in the presence of inactivated human blood serum. Further studies are needed to confirm its mode of action. (C) 2019 Elsevier Masson SAS. All rights reserved.Peer reviewe

Rapid evolution of reduced susceptibility against a balanced dual targeting antibiotic through stepping-stone mutations

Multi-targeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets are generally considered as a promising therapeutic strategy against resistance evolution. The rationale for this theory is that multi-targeting antibiotics demand the simultaneous acquisition of multiple mutations at their respective target genes to achieve significant resistance. The theory presumes that individual mutations provide little or no benefit to the bacterial host. Here we propose that such individual, stepping-stone mutations can be prevalent in clinical bacterial isolates, as they provide significant resistance to other antimicrobial agents. To test this possibility, we focused on gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an over 2000-fold reduction in susceptibility, while individually none of these mutations affect resistance significantly. Alarmingly, strains with decreased susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed gyrase inhibitor, co-selected for reduced susceptibility against gepotidacin. We conclude that extensive antibiotic usage could select for mutations that serve as stepping-stones towards resistance against antimicrobial compounds still under development. Our research indicates that even balanced multi-targeting antibiotics are prone to resistance evolution

Second generation 4,5,6,7-tetrahydrobenzo[d]thiazoles as novel DNA gyrase inhibitors

Aim: DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel antibacterial drugs. Results: Building from our first generation of 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase inhibitors, we designed and prepared an optimized series of analogs that show improved inhibition of DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli, with IC50 values in the nanomolar range. Importantly, these inhibitors also show improved antibacterial activity against Gram-positive strains. Conclusion: The most promising inhibitor, 29, is active against Enterococcus faecalis, Enterococcus faecium and S. aureus wild-type and resistant strains, with minimum inhibitory concentrations between 4 and 8 mu g/ml, which represents good starting point for development of novel antibacterials. Graphical abstractPeer reviewe

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase. offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any in vitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625 mg/mL) and Gram-negative pathogens (MICs: range, 1-2 mg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance. (C) 2021 Elsevier Masson SAS. All rights reserved.Peer reviewe

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology

Dual Escherichia coli DNA Gyrase A and B Inhibitors with Antibacterial Activity.

The emergence of multidrug-resistant bacteria is a global health threat necessitating the discovery of new antibacterials and novel strategies for fighting bacterial infections. We report first-in-class DNA gyrase B (GyrB) inhibitor/ciprofloxacin hybrids that display antibacterial activity against Escherichia coli. Whereas DNA gyrase ATPase inhibition experiments, DNA gyrase supercoiling assays, and in vitro antibacterial assays suggest binding of the hybrids to the E. coli GyrA and GyrB subunits, an interaction with the GyrA fluoroquinolone-binding site seems to be solely responsible for their antibacterial activity. Our results provide a foundation for a new concept of facilitating entry of nonpermeating GyrB inhibitors into bacteria by conjugation with ciprofloxacin, a highly permeable GyrA inhibitor. A hybrid molecule containing GyrA and GyrB inhibitor parts entering the bacterial cell would then elicit a strong antibacterial effect by inhibition of both the GyrA and GyrB subunits of DNA gyrase and potentially slow bacterial resistance development