Modification of Azithromycin to Mitigate its Arrest of Autophagy

Aims/hypothesis Autophagy plays a critical role in the survival and microbial clearance functions of airway epithelial cells ( AEC ). Contrary to the known roles of azithromycin ( AZM ) in promoting microbial clearance, our preliminary evidence suggests that AZM inhibits autophagy in mammalian cells - an effect that could potentially promote bacterial residency in chronic respiratory diseases. Here we investigate the structure-activity relationship of AZM vs an AZM derivative ( AZM-[O] ), to mitigate the off-target arrest of autophagy. Method Participant-derived COPD vs control airway biopsies were assessed for intrinsic defects in autophagy gene activity via qRT-PCR array. For in vitro studies, the 16HBE14o-AEC line and Western blot were used to assess macrolide vs autophagy structure-activity relationships, and autophagic flux by quantifying the protein abundance of LC3B-II vs Sequestosome-1. Subsequent assessments of antimicrobial activity were conducted using an E coli model and the minimum inhibitory concentration method. Immunomodulatory outcomes were assessed by quantifying the secretion of IL-6 in an LPS-stimulated THP-1 macrophage model. Results Signatures of transcript-abundance showed an overall reduction in autophagy gene activity in COPD vs control biopsies. AZM significantly inhibited autophagic flux, evidenced by the accumulation of LC3B-II and Sequestosome-1, compared to its precursors and other macrolides including roxithromycin and clarithromycin. Notably, oxidation of AZM to produce AZM-[O] significantly alleviated this inhibitory effect on autophagy without completely preserving its antimicrobial and immunomodulatory functions. Conclusion The potent off-target block of autophagic flux demonstrated by AZM can be negated using a medicinal chemistry approach. We are currently investigating the AZM-protein interactome and further AZM derivatives that preserve the immunomodulatory function of the parent molecule, to provide a new therapeutic option for chronic respiratory disease and to safeguard the efficacy of AZM. ### Competing Interest Statement The authors have declared no competing interest.