Sapula, S. A. | bioRxiv Science

bioRxiv2026

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 h 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 ug/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 ug/ml, where as D1 and D2 were 1.0 and 8.0 ug/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.

cell biology↗

bioRxiv2024

Modification of Azithromycin to Mitigate its Arrest of Autophagy

Aims/hypothesisAutophagy plays a critical role in the survival and microbial clearance function of tissues that encounter the environment, such as airway epithelial cells (AECs). Contrary to the known roles of azithromycin (AZM) in promoting microbial clearance, our evidence shows that AZM is a potent inhibitor of autophagy - an effect consistent with bacterial residency. Here we investigate the structure-activity relationship of AZM vs other macrolides and AZM-3-N-oxide (AZM-[O]), to mitigate the off-target arrest of autophagy. MethodAZM-[O] was synthesised in-house via selective oxidation of the desosamine amine of AZM. Human peripheral blood mononuclear cells (PBMC) were used to assess autophagy ex vivo in an organotypic manner via transmission electron microscopy and Western blot analysis. For in vitro studies, the 16HBE14o-AEC line and Western blot was 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 the micro-broth dilution method. Immunomodulatory outcomes were assessed by quantifying the secretion of IL-6 in a lipopolysaccharide PMA-stimulated THP-1 macrophage model. ResultsAZM significantly inhibited autophagic flux in both the ex vivo PBMC and in vitro 16HBE14o-AEC models, evidenced by the accumulation of autophagosome-related vacuoles and LC3B-II and Sequestosome-1 protein, 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, but without completely preserving its antimicrobial and immunomodulatory functions. Conclusion/DirectionsWe show for the first time that AZM inhibits PBMC autophagy in human blood ex vivo and there is a high probability this phenomenon occurs in the clinical setting. Importantly, chemical modification of AZM to generate AZM-[O] substantially alleviated this effect, consistent with altered ionisation properties. We are now assessing clinically derived blood samples in participants treated with AZM, defining the AZM-mammalian protein interactome and developing further AZM derivatives that preserve immunomodulatory activity while minimising disruption of autophagy.

biochemistry↗