Biology subjects

Kopp, B. T.

Publications and source records attributed to Kopp, B. T..

3 recordsLinked to original sources

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↗

bioRxiv2025

CFTR mutation leads to intrinsic dysfunction in neutrophils from people with Cystic Fibrosis

Cystic fibrosis (CF), a common genetic disease, is caused by a defective CF-transmembrane conductance regulator (CFTR). People with CF (pwCF) are prone to develop infections by opportunistic pathogens, including Burkholderia cenocepacia, leading to chronic inflammation and lung function loss. Neutrophils, the most abundant cells in the chronically inflamed lungs of pwCF, release granular proteins and oxidative products that contribute to tissue damage. The CFTR modulators are a new treatment for pwCF aiming to correct the subcellular location and function of the CFTR ion channel. The triple modulator combination of Elexacaftor, Tezacaftor, and Ivacaftor (ETI) or Trikafta(R) has significantly improved clinical symptoms and overall provided a better quality of life for pwCF. The mechanism by which the CFTR modulators help to restore the antimicrobial functions of neutrophils is unknown. The present study demonstrates that neutrophils functionally express CFTR and reveals how ETI modifies subcellular CFTR trafficking in CF neutrophils. In addition, ETI treatment reduces intracellular chloride levels in human neutrophils, indicating activation of CFTR-dependent chloride efflux (outflow). Finally, ETI treatment also reestablished the intracellular antimicrobial killing of CF neutrophils by potentiating NADPH oxidase activity and producing Neutrophil Extracellular Traps (NETs). Together, our findings suggest that CFTR has an essential role in controlling neutrophil functions and that the CFTR modulators improve the health of pwCF by restoring the antimicrobial functions of CF neutrophils. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/656500v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@8469b5org.highwire.dtl.DTLVardef@1380609org.highwire.dtl.DTLVardef@1145117org.highwire.dtl.DTLVardef@d7c0a2_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO (A) The F508del defective CFTR protein cannot reach the plasma membrane in CF neutrophils, which increases the intracellular concentrations of Cl- ions, and allows other ions to be internalized, including Na+ and Ca2+. This ionic imbalance affects the NADPH oxidase, leading to a reduced preactivation response and consequently impacting NADPH oxidase-dependent antimicrobial mechanisms, including intracellular antimicrobial killing and NETosis. (B) Treating with ETI restores the CFTR expression in the plasma membrane of CF neutrophils, increasing the Cl- efflux and regulating the intracellular levels of Na+ and Ca2+, leading to correcting the NADPH oxidase, which results in potentiating the intracellular antimicrobial killing and NETosis. BiorenderTM tools generated the images. C_FIG

immunology↗

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↗