Aim: We have shown that Azithromycin (AZM) blocks airway epithelial cell autophagy. This is contrary to autophagy and AZM?s role of clearing intracellular microbes, and hence may promote bacterial residence and tolerance.

Hypothesis: Altering the charge state of AZM or cotreatment with promoters of autophagy counters AZM?s off-target block of autophagy.

Method: The 16HBE14o- line was used to model/assess autophagy. Synthetic chemistry was used to produce an oxidised form of AZM, termed ?-AZM. In parallel experiments, the autophagy promoters Ku-0063794 (KU) or Torin-1 were co-treated with conventional AZM. Structure-activity analyses were then performed by quantifying the abundance of LC3-II (indispensable for autophagy) vs Sequestosome (decreases when autophagy is promoted), to generate a readout for autophagic-flux using Western blot analysis.

Results: KU (10 uM) or Torin-1 (5-10 nM) significantly released AZM-based (50 ug/mL) block of autophagy (P<0.01). 9a-NBD-AZM (a fluorescent AZM analogue) loaded into 16HBE14o- cells exposed to KU, showed this was achieved by the cells synthesising further autophagosomes. ?-AZM significantly mitigated blockade of autophagic flux vs conventional AZM (P<0.001). However, ?-AZM exhibited a lower bacteriostatic effect in an E coli model and reduced anti-inflammatory properties, as determined using LPS stimulation and readouts for IL-6.

Conclusion: We provide proof of concept that AZM-mediated autophagic blockade can be mitigated with autophagy promoters and by modifying AZM structure. We are currently investigating further AZM derivatives that conserve the antibiotic and anti-inflammatory functions of the parent molecule, to safeguard AZM as a frontline option for respiratory clinicians.