Quarrington, R. D.; Sapula, S. A.; Lester, S. E.; Matthew, M. M.; Kos, V. M.; Kopp, B. T.; Jersmann, H. P.; Blencowe, A.; Roscioli, E.

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 using an 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 p62/SQSTM1, compared to its precursors and other macrolides including roxithromycin and clarithromycin. Notably, oxidation of AZM to AZM-[O] significantly alleviated this inhibitory effect on autophagy without completely preserving its antimicrobial and immunomodulatory functions. Conclusion: The off-target and potent 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.