Cystobactamid off-target profiling reveals favorable safety, superoxide reduction, and SCARB1 inhibition in eukaryotes

Abstract

Antimicrobial resistance (AMR) is escalating globally at an alarming rate, posing a severe threat to public health and modern medicine. A substantial strategy to combat AMR and ensure safe and efficient antimicrobial therapies in the future is the development of antimicrobial agents based on novel scaffolds exerting an innovative mode of action. The antibacterial class of cystobactamids, natural products targeting the bacterial gyrase and topoisomerase IV, have such a novel scaffold, and their synthetic derivatives exhibit broad-spectrum antibiotic activity. Herein, as part of our de-risking strategy, we set out to study and characterize potential biological effects on eukaryotic cells. A comprehensive toxicological in vitro profiling focusing on general cytotoxicity, genotoxicity and mitochondrial toxicity is provided along with in vivo experiments to characterize potential developmental, cardio- and hepatotoxicity in zebrafish embryos. Results from these investigations support good safety and a presumably high therapeutic index of cystobactamids, even suggesting potential protective properties against oxidative cell stress. Furthermore, in vitro metabolism studies unveiled glucuronidation and amide bond hydrolysis as the main pathways of biotransformation in hepatocytes. Metabolic stability of cystobactamids was substantially improved by co-treatment with the CYP3A/OATP1B inhibitor cobicistat. In addition, eukaryotic off-targets of cystobactamids were studied on the molecular level employing affinity-based protein profiling and revealing scavenger receptor class B member 1 (SCARB1) as primary eukaryotic target protein. Indeed, functional inhibition of SCARB1 by cystobactamids was shown and effectively prevented hepatitis C virus pseudoparticles (HCVpp) from cell entry, thus confirming SCARB1 inhibition as novel target of the class.