Dendrimer-based selective autophagy-induction rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis

Abstract

BackgroundCystic Fibrosis (CF) is a genetic disorder caused by mutation(s) in the CF-transmembrane conductance regulator (Cftr) gene. The most common mutation, ΔF508, leads to accumulation of defective-CFTR protein in aggresome-bodies. Additionally, Pseudomonas aeruginosa (Pa), a common CF pathogen, exacerbates obstructive CF lung pathology. In the present study, we aimed to develop and test a novel strategy to improve the bioavailability and potentially achieve targeted drug delivery of cysteamine, a potent autophagy-inducing drug with anti-bacterial properties, by developing a dendrimer (PAMAM-DEN)-based cysteamine analogue.ResultsWe first evaluated the effect of dendrimer-based cysteamine analogue (PAMAM-DENCYS) on the intrinsic autophagy response in IB3-1 cells and observed a significant reduction in Ub-RFP and LC3-GFP co-localization (aggresome-bodies) by PAMAM-DENCYS treatment as compared to plain dendrimer (PAMAM-DEN) control. Next, we observed that PAMAM-DENCYS treatment shows a modest rescue of ΔF508-CFTR as the C-form. Moreover, immunofluorescence microscopy of HEK-293 cells transfected with ΔF508-CFTR-GFP showed that PAMAM-DENCYS is able to rescue the misfolded-ΔF508-CFTR from aggresome-bodies by inducing its trafficking to the plasma membrane. We further verified these results by flow cytometry and observed significant (p<0.05; PAMAM-DEN vs. PAMAM-DENCYS) rescue of membrane-ΔF508-CFTR with PAMAM-DENCYS treatment using non-permeabilized IB3-1 cells immunostained for CFTR. Finally, we assessed the autophagy-mediated bacterial clearance potential of PAMAM-DENCYS by treating IB3-1 cells infected with PA01-GFP, and observed a significant (p<0.01; PAMAM-DEN vs. PAMAM-DENCYS) decrease in intracellular bacterial counts by immunofluorescence microscopy and flow cytometry. Also, PAMAM-DENCYS treatment significantly inhibits the growth of PA01-GFP bacteria and demonstrates potent mucolytic properties.ConclusionsWe demonstrate here the efficacy of dendrimer-based autophagy-induction in preventing sequestration of ΔF508-CFTR to aggresome-bodies while promoting its trafficking to the plasma membrane. Moreover, PAMAM-DENCYS decreases Pa infection and growth, while showing mucolytic properties, suggesting its potential in rescuing Pa-induced ΔF508-CF lung disease that warrants further investigation in CF murine model.

Silencing of the Hsp70-specific nucleotide-exchange factor BAG3 corrects the F508del-CFTR variant by restoring autophagy

“…pathways at the bench and bedside (58,(62)(63)(64)(65)(66)(67)(68)(69). Our data reveal that disrupting the delivery of CFTR disease-associated variants to both the UPS, via silencing of BAG1, and autophagosome, via the silencing of BAG3, corrects the trafficking defect associated with F508del-CFTR, leading to restoration of cellsurface activity.…”

“…Previously published efforts have emphasized the importance of autophagy in rescue of CFTR (58,(62)(63)(64)(65)(66)(67)(68)(69). Given the central role of BAG3 in the management of autophagy pathways for multiple targets (70 -74), we have demonstrated that reducing the cellular expression level of BAG3 generates a proteostatic environment that provides both chaperoning activity of F508del-CFTR, leading to improved biogenesis, and restoration of cell surface chloride channel activity.…”

Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies

“…The cysteamine drug (Lynovex ® , Novabiotics), an oral mucoactive, antibiofilm, and antibacterial agent, has passed the phase IIb clinical trial (NCT03000348) for use in the treatment of acute, infectious CF exacerbations [213]. PAMAM dendrimers were modified as cysteamine-like structure dendrimers (PAMAM-DEN CYS ), to obtain the antibiofilm activity of cysteamine [214]. PAMAM-DEN CYS demonstrated enhanced penetration across the thick mucus layer, thus, reducing biofilm formation, and the infection associated with P. aeruginosa [214].…”

Drug efficacy and toxicity prediction: an innovative application of transcriptomic data

“…Lumacaftor/ivacaftor for CF is a drug combination representative of restorative drugs. The majority of CF is caused by the deletion of F508 (ΔF508) in both alleles of the CFTR gene (Brockman et al 2017;Esposito et al 2016;Faure et al 2016). The ΔF508 CFTR proteins cannot be folded properly in ER lumen and are mostly degraded after failing to be exported to the cell membrane to perform its ion channel function (Fraser-Pitt and O'Neil 2015).…”