Azithromycin Derivatives to Mitigate Off-Target Inhibition of Autophagy and Retain Beneficial Host Directed Effects

Azithromycin (AZM) is central for the treatment of chronic respiratory diseases (CRD) but has divergent off-target effects. We synthesised AZM Derivatives 1 and 2 (D1/D2) that were predicted to permit autophagy and preserve AZM’s anti-inflammatory effect. The 16HBE14o- airway epithelial cell model was exposed to AZM, D1 and D2 for 16 hr and assessed for autophagy flux via LC3B-II:p62/SQSTM1 abundance (Western blot). Necrosis was quantified via lactate dehydrogenase release. Inflammation (IL-6 secretion) was assessed in the THP-1 macrophage model exposed to 10 ng/mL lipopolysaccharide vs co-treatment with AZM and the derivatives for 18 h. AZM-derivative antibacterial activity (vs AZM) was determined via the minimum inhibition concentration (MIC) method using methicillin sensitive Staphylococcus aureus (MSSA) . Autophagy (LC3B-II and p62/SQSTM1 abundance) was not altered by the two derivatives and was indistinguishable from the control exposure (P> 0.05 for D1 and D2, each 10 and 50 µg/mL, vs control). D2 elicited a significant decrease in LPS-induced IL-6 secretion vs the LPS-only exposure (58.22 pg/ml, n=3, 95% ± CI [6.521 – 109.9]). Importantly, D2 caused a similar reduction in LPS-induced IL-6 secretion, as observed for AZM (-10.30 pg/mL, n=3, 95% CI [-62.00 to 41.39]). The MIC of AZM for MSSA growth was 0.5 µg/ml, where as D1 and D2 were 1.0 and 8.0 µg/mL, respectively (P<0.05). We show for the first time that AZM can be redesigned to mitigate its potent arrest of autophagy while preserving its anti-inflammatory activity, to counter the generation of further AZM resistant strains.