Structure, function, and inhibition of bacterial Arr ADP-ribosyltransferases

Abstract

Antibiotic resistance is a significant challenge to public health and requires innovative strategies to overcome bacterial infections resistant to multiple drugs. Bacterial species have developed various antibiotic resistance mechanisms, including producing enzymes like Arrs. These bacterial ADP-ribosyltransferases modify the rifamycin antibiotic through ADP-ribosylation, causing its inactivation by inhibiting its ability to bind to the β subunit of RNA polymerase. This study aimed to identify novel inhibitors targeting ADP-ribosyltransferases (Arrs) in Mycobacterium smegmatis (MsArr), Pseudomonas aeruginosa (PaArr), Stenotrophomonas maltophilia (SmaArr), and Mycobacterium abscessus (MaArr). Successful cloning, expression, and enzymatic confirmation of Arrs were achieved. Potential inhibitors were discovered through screening of transferase-targeted inhibitor libraries with a fluorescence assay measuring NAD+ consumption. Best inhibitors had nanomolar IC50 values and stabilized Arrs in a thermal shift assay in NanoDSF analysis. Furthermore, structural elucidation was achieved through X-ray crystallography of PaArr and MsArr together with inhibitors. Notably, the crystal structure of PaArr revealed unique structural differences, distinguishing it from MsArr. These findings might offer promising paths for developing effective approaches to combat antibiotic resistance. Additionally, targeting Arrs in vivo may shed light on the diverse functional roles of Arrs that are currently poorly understood, leading to a more comprehensive understanding of their potential impact.