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.

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.

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.

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.

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.