Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.

Antimicrobial activity of POL7306 tested against clinical isolates of Gram-negative bacteria collected worldwide.

BACKGROUND: POL7306 belongs to a new class of peptidomimetic outer-membrane-protein-targeting antibiotics with a novel mechanism of action. POL7306 is in development for the treatment of infections caused by antimicrobial-resistant Gram-negative bacteria and has demonstrated low cytotoxicity and nephrotoxicity. METHODS: A total of 891 isolates were collected by the SENTRY Antimicrobial Surveillance Program from 134 medical centres in Europe (n = 424; 41 centres in 18 nations), the USA (n = 411 isolates from 67 centres), the Asia-Pacific region (n = 24; 15 centres in 6 nations) and Latin America (n = 32; 11 centres in 9 nations) and included 558 Enterobacterales, 310 non-fermenters and 23 fastidious organisms. Susceptibility testing was performed using the reference broth microdilution method and the medium was supplemented with 0.002% polysorbate-80 for testing POL7306. Resistant subsets were characterized by WGS. RESULTS: POL7306 demonstrated potent in vitro activity against Enterobacterales [including carbapenem-resistant (MIC50/90, 0.06/0.25 mg/L), ESBL-producing (MIC50/90, 0.06/0.12 mg/L), KPC-producing (MIC50/90, 0.12/0.25 mg/L), MBL-producing (MIC50/90, 0.06/0.25 mg/L), colistin-non-susceptible, mcr-negative (MIC50/90, 0.5/2 mg/L) and mcr-positive (MIC50/90, 0.12/0.25 mg/L) Enterobacterales], Pseudomonas aeruginosa (MIC50/90, 0.25/0.25 mg/L), Acinetobacter baumannii (MIC50/90, 0.06/0.12 mg/L) and Stenotrophomonas maltophilia (MIC50/90, 0.06/0.25 mg/L). CONCLUSIONS: POL7306 demonstrated potent activity against a large collection of Gram-negative organisms collected worldwide that included colistin-resistant, XDR and ESBL- and carbapenemase-producing isolates for which there are currently limited treatment options.

Different Resistance Mechanisms for Cadazolid and Linezolid in Clostridium difficile Found by Whole-Genome Sequencing Analysis.

Cadazolid (CDZ) is a new antibiotic currently in clinical development for the treatment of Clostridium difficile infections. CDZ interferes with the bacterial protein synthesis machinery. The aim of the present study was to identify resistance mechanisms for CDZ and compare the results to those obtained for linezolid (LZD) in C. difficile by whole-genome sequencing (WGS) of strains generated by in vitro passages and to those obtained for LZD-resistant clinical isolates. Clones of C. difficile 630 selected with CDZ during 46 passages had a maximally 4-fold increase in CDZ MIC, while the LZD MIC for clones selected with LZD increased up to 16-fold. CDZ cross-resistance with LZD was maximally 4-fold, and no cross-resistance with other antibiotics tested was observed. Our data suggest that there are different resistance mechanisms for CDZ and LZD in C. difficile Mutations after passages with CDZ were found in rplD (ribosomal protein L4) as well as in tra and rmt, whereas similar experiments with LZD showed mutations in rplC (ribosomal protein L3), reg, and tpr, indicating different resistance mechanisms. Although high degrees of variation between the sequenced genomes of the clinical isolates were observed, the same mutation in rplC was found in two clinical isolates with high LZD MICs. No mutations were found in the 23S rRNA genes, and attempts to isolate the cfr gene from resistant clinical isolates were unsuccessful. Analysis of 50% inhibitory concentrations (IC50s) determined in in vitro transcription/translation assays performed with C. difficile cell extracts from passaged clones correlated well with the MIC values for all antibiotics tested, indicating that the ribosomal mutations are causing the resistant phenotype.

Cadazolid Does Not Promote Intestinal Colonization of Vancomycin-Resistant Enterococci in Mice.

The promotion of colonization with vancomycin-resistant enterococci (VRE) is one potential side effect during treatment of Clostridium difficile-associated diarrhea (CDAD), resulting from disturbances in gut microbiota. Cadazolid (CDZ) is an investigational antibiotic with potent in vitro activity against C. difficile and against VRE and is currently in clinical development for the treatment of CDAD. We report that CDZ treatment did not lead to intestinal VRE overgrowth in mice.

Investigations of the mode of action and resistance development of cadazolid, a new antibiotic for treatment of Clostridium difficile infections.

Cadazolid is a new oxazolidinone-type antibiotic currently in clinical development for the treatment of Clostridium difficile-associated diarrhea. Here, we report investigations on the mode of action and the propensity for spontaneous resistance development in C. difficile strains. Macromolecular labeling experiments indicated that cadazolid acts as a potent inhibitor of protein synthesis, while inhibition of DNA synthesis was also observed, albeit only at substantially higher concentrations of the drug. Strong inhibition of protein synthesis was also obtained in strains resistant to linezolid, in agreement with low MICs against such strains. Inhibition of protein synthesis was confirmed in coupled transcription/translation assays using extracts from different C. difficile strains, including strains resistant to linezolid, while inhibitory effects in DNA topoisomerase assays were weak or not detectable under the assay conditions. Spontaneous resistance frequencies of cadazolid were low in all strains tested (generally <10(-10) at 2× to 4× the MIC), and in multiple-passage experiments (up to 13 passages) MICs did not significantly increase. Furthermore, no cross-resistance was observed, as cadazolid retained potent activity against strains resistant or nonsusceptible to linezolid, fluoroquinolones, and the new antibiotic fidaxomicin. In conclusion, the data presented here indicate that cadazolid acts primarily by inhibition of protein synthesis, with weak inhibition of DNA synthesis as a potential second mode of action, and suggest a low potential for spontaneous resistance development.

In vitro and in vivo antibacterial evaluation of cadazolid, a new antibiotic for treatment of Clostridium difficile infections.

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 µg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.

Discovery and Structure-Activity Relationship of Cadazolid: A First-In-Class Quinoxolidinone Antibiotic for the Treatment of Clostridioides difficile Infection.

Clostridioides difficile (C. difficile) is one of the leading causes of healthcare-associated infections worldwide. The increasing incidence of strains resistant to currently available therapies highlights the need for alternative treatment options with a novel mode of action. Oxazolidinones that are connected to a quinolone moiety with a pyrrolidine linker, such as compound 1, are reported to exhibit potent broadspectrum antibacterial activity. In an effort to optimize this class of compounds for the treatment of C. difficile infection (CDI), we have identified cadazolid (9), a first-in-class quinoxolidinone antibiotic, which is a potent inhibitor of C. difficile protein synthesis. In order to achieve narrow-spectrum coverage of clinically most relevant strains without affecting the gut microbiota, an emphasis was placed on abolishing activity against commensals of the intestinal microbiome while retaining good coverage of pathogenic C. difficile, including hypervirulent and epidemic strains.

Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae.

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.

Structure-guided design, synthesis and biological evaluation of novel DNA ligase inhibitors with in vitro and in vivo anti-staphylococcal activity.

A series of 2-amino-[1,8]-naphthyridine-3-carboxamides (ANCs) with potent inhibition of bacterial NAD(+)-dependent DNA ligases (LigAs) evolved from a 2,4-diaminopteridine derivative discovered by HTS. The design was guided by several highly resolved X-ray structures of our inhibitors in complex with either Streptococcus pneumoniae or Escherichia coli LigA. The structure-activity-relationship based on the ANC scaffold is discussed. The in-depth characterization of 2-amino-6-bromo-7-(trifluoromethyl)-[1,8]-naphthyridine-3-carboxamide, which displayed promising in vitro (MIC Staphylococcus aureus 1 mg/L) and in vivo anti-staphylococcal activity, is presented.

Dimers of nostocarboline with potent antibacterial activity.

OBJECTIVES: In this study, the in vitro antimicrobial activity and spectrum of new dimeric compounds derived from the cyanobacterial alkaloid nostocarboline were investigated. The mechanism of action and selectivity to bacteria were studied and compared to the cationic antiseptic chlorhexidine. METHODS: Minimal inhibitory concentrations were determined against clinical isolates and against a panel of microbial reference strains using the CLSI microdilution method. Bacterial membrane damage was addressed by measuring ATP leakage and the mode of action was investigated in Escherichia coli reporter strains. Selectivity was tested by a cytotoxicity assay using MTS. RESULTS: The antimicrobial potency of dimers varied with length of the hydrophobic linker. The most potent compounds, NCD9 and NCD10, had a C10 and C12 linker, respectively, and showed strong activity against Gram-positive bacteria, notably methicillin-resistant Staphylococcus aureus strains. Similar to chlorhexidine, these compounds showed a rapid concentration-dependent bactericidal effect, which correlated with membrane damage as indicated by ATP leakage. NCD9, in contrast to NCD10 and chlorhexidine, lacked activity against yeast strains and showed low cytotoxicity in CHO cells indicating a high degree of selectivity. In E. coli reporter strains, NCD9 induced the DegP response pathway as well as the SOS response, suggesting interaction with both the cell envelope and DNA metabolism. CONCLUSIONS: The results presented in this report indicate the potential of this new class of cationic antimicrobial compounds for the design of potent and selective antibacterials with low cytotoxicity.

Identification of Genes Required for Resistance to Peptidomimetic Antibiotics by Transposon Sequencing.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections. Due to its high intrinsic and adaptive resistance to antibiotics, infections caused by this organism are difficult to treat and new therapeutic options are urgently needed. Novel peptidomimetic antibiotics that target outer membrane (OM) proteins have shown great promise for the treatment of P. aeruginosa infections. Here, we have performed genome-wide mutant fitness profiling using transposon sequencing (Tn-Seq) to identify resistance determinants against the recently described peptidomimetics L27-11, compounds 3 and 4, as well as polymyxin B2 (PMB) and colistin (COL). We identified a set of 13 core genes that affected resistance to all tested antibiotics, many of which encode enzymes involved in the modification of the lipopolysaccharide (LPS) or control their expression. We also identified fitness determinants that are specific for antibiotics with similar structures that may indicate differences in their modes of action. These results provide new insights into resistance mechanisms against these peptide antibiotics, which will be important for future clinical development and efforts to further improve their potency.

Optimization of LpxC Inhibitor Lead Compounds Focusing on Efficacy and Formulation for High Dose Intravenous Administration.

LpxC inhibitors were optimized starting from lead compounds with limited efficacy and solubility and with the goal to provide new options for the treatment of serious infections caused by Gram-negative pathogens in hospital settings. To enable the development of an aqueous formulation for intravenous administration of the drug at high dose, improvements in both solubility and antibacterial activity in vivo were prioritized early on. This lead optimization program resulted in the discovery of compounds such as 13 and 30, which exhibited high solubility and potent efficacy against Gram-negative pathogens in animal infection models.

Structural basis of translation inhibition by cadazolid, a novel quinoxolidinone antibiotic.

Oxazolidinones are synthetic antibiotics used for treatment of infections caused by Gram-positive bacteria. They target the bacterial protein synthesis machinery by binding to the peptidyl transferase centre (PTC) of the ribosome and interfering with the peptidyl transferase reaction. Cadazolid is the first member of quinoxolidinone antibiotics, which are characterized by combining the pharmacophores of oxazolidinones and fluoroquinolones, and it is evaluated for treatment of Clostridium difficile gastrointestinal infections that frequently occur in hospitalized patients. In vitro protein synthesis inhibition by cadazolid was shown in Escherichia coli and Staphylococcus aureus, including an isolate resistant against linezolid, the prototypical oxazolidinone antibiotic. To better understand the mechanism of inhibition, we determined a 3.0 Å cryo-electron microscopy structure of cadazolid bound to the E. coli ribosome in complex with mRNA and initiator tRNA. Here we show that cadazolid binds with its oxazolidinone moiety in a binding pocket in close vicinity of the PTC as observed previously for linezolid, and that it extends its unique fluoroquinolone moiety towards the A-site of the PTC. In this position, the drug inhibits protein synthesis by interfering with the binding of tRNA to the A-site, suggesting that its chemical features also can enable the inhibition of linezolid-resistant strains.

Time-kill kinetics of cadazolid and comparator antibacterial agents against different ribotypes of Clostridium difficile.

PURPOSE: Clostridium difficile infection (CDI) is an increasing cause of nosocomial diarrhoea worldwide, which has been partly attributed to the emergence of hypervirulent strains including C. difficile BI/NAP1/ribotype 027 and BK/NAP7/ribotype 078. Cadazolid is a new antibiotic currently in late-stage clinical studies for the treatment of CDI. The present study evaluated the in vitro bactericidal effect of cadazolid and comparator antibiotics against four C. difficile strains. The data demonstrate the potent and bactericidal activity of cadazolid against different ribotypes of C. difficile. METHODOLOGY: MICs for test antibiotics were determined in brain- heart infusion-supplemented broth (BHIS) containing 5 g l-1 yeast extract and 0.025 % (w/v) l-cysteine. Time-kill kinetics to investigate the rate of killing of each antibiotic at sub- and supra-MIC concentrations were performed at concentrations of 0.5, 1, 2, 4, 8 or 16× the MIC of cadazolid, vancomycin and fidaxomicin at intervals over a 48 h period.Results/key findings. Cadazolid-mediated killing of C. difficile was faster and occurred at lower concentrations than observed for vancomycin, while potency and killing was largely comparable to those observed for fidaxomicin. Notably, cadazolid also displayed a potent bactericidal effect against fluoroquinolone-resistant hypervirulent ribotype 027 and 078 strains. C. difficile spore formation was largely inhibited by all three antibiotics at concentrations >1× MIC; however, none were able to eliminate spores effectively, which were present at the start of the experiment. CONCLUSION: The data presented here demonstrate the potent in vitro bactericidal activity of cadazolid against different ribotypes of C. difficile, although on a limited set of strains.

Synthesis and Characterization of Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors with Antibacterial Activity against Gram-Negative Bacteria.

There is an urgent unmet medical need for novel antibiotics that are effective against a broad range of bacterial species, especially multidrug resistant ones. Tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent activity against Gram-positive pathogens and no target-mediated cross-resistance with fluoroquinolones. We report our research efforts aimed at expanding the antibacterial spectrum of this class of molecules toward difficult-to-treat Gram-negative pathogens. Physicochemical properties (polarity and basicity) were considered to guide the design process. Dibasic tetrahydropyran-based compounds such as 6 and 21 are potent inhibitors of both DNA gyrase and topoisomerase IV, displaying antibacterial activities against Gram-positive and Gram-negative pathogens (Staphylococcus aureus, Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). Compounds 6 and 21 are efficacious in clinically relevant murine infection models.

Potent algicides based on the cyanobacterial alkaloid nostocarboline.

[structure: see text] Nostocarboline and seven derivatives were prepared and displayed minimal inhibitory concentration (MIC) values >or=100 nM against the growth of Microcystis aeruginosa PCC 7806, Synechococcus PCC 6911, and Kirchneriella contorta SAG 11.81, probably via the inhibition of photosynthesis. The natural product hybrid nostocarboline/ciprofloxacin displayed additional antibacterial activity against several Gram-negative bacteria (MICs >or=0.7 microM). Nostocarboline can thus be considered a potent, selective, readily available, natural algicide.

Novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram positive activity and improved safety profile.

Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K(+) channels and hNaV1.5 Na(+) channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.

Novel dihydrofolate reductase inhibitors. Structure-based versus diversity-based library design and high-throughput synthesis and screening.

Novel 2,4-diaminopyrimidines bearing N,N-disubstituted aminomethyl residues at the 5-position were designed as dihydrofolate reductase (DHFR) inhibitors. These compounds were obtained by treatment of 1-[(2,4-diamino-5-pyrimidinyl)methyl]pyridinium bromide with secondary amines in a polar solvent and in the presence of triethylamine at room temperature. The procedure was found to be very efficient and suitable for application in high-throughput synthesis. In addition, we found that high-throughput screening for enzymatic and in vitro antibacterial activity could be performed on crude reaction mixtures, thus avoiding any purification step. Over 1200 proprietary secondary amines were selected for high-throughput synthesis, based on structural and diversity-related criteria, and the resulting products were submitted to high-throughput screening. A greater number of hits, and significantly more active compounds, were obtained through structure-based library design than through diversity-based library design. Different classes of inhibitors of DHFR were identified in this way, including compounds derived from di-, tri-, and tetracyclic amines. In general, these products showed high activity against the enzymes derived from both TMP-sensitive and TMP-resistant Streptococcus pneumoniae. Some compounds possessed appreciable selectivity for the bacterial over the human enzyme, whereas other compounds were not at all selective. In most cases, active enzyme inhibitors also displayed antibacterial activity.

Design, synthesis, and characterization of novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram-positive activity.

There is an urgent need for new antibacterial drugs that are effective against infections caused by multidrug-resistant pathogens. Novel nonfluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) have the potential to become such drugs because they display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. Bacterial topoisomerase inhibitors that are built on a tetrahydropyran ring linked to a bicyclic aromatic moiety through a syn-diol linker show potent anti-Gram-positive activity, covering isolates with clinically relevant resistance phenotypes. For instance, analog 49c was found to be a dual DNA gyrase-topoisomerase IV inhibitor, with broad antibacterial activity and low propensity for spontaneous resistance development, but suffered from high hERG K(+) channel block. On the other hand, analog 49e displayed lower hERG K(+) channel block while retaining potent in vitro antibacterial activity and acceptable frequency for resistance development. Furthermore, analog 49e showed moderate clearance in rat and promising in vivo efficacy against Staphylococcus aureus in a murine infection model.