163 | SIX-2G as a novel compound to attenuate airway inflammation in cystic fibrosis through modulation of molecular chaperones

Giuseppa D’Amico¹, Federica Scalia², Maria Antonella Augello¹, Giusi Alberti¹, Roberta Bivacqua³, Marilia Barreca³, Rosario Barone¹, Francesco Cappello¹, Paola Barraja³, Alessandra Montalbano³, Celeste Caruso Bavisotto¹ | ¹Department of Biomedicine, Neuroscience and Advanced Diagnostics BIND, University of Palermo, Italy; ²Department of Medicine and Surgery, Kore University of Enna, Italy; ³Department of Biological, Chemical, Pharmaceutical Sciences and Technologies STEBICEF, University of Palermo, Italy.

Cystic fibrosis (CF) is characterized by chronic airway inflammation, which results in progressive lung damage, with dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) as key molecular factor underlying these pathological processes [1]. Increasing evidence suggests that dysregulated proteostasis and aberrant chaperone activity contribute not only to defective CFTR processing but also to persistent inflammatory signaling in airway epithelial cells [2]. In this context, targeting chaperone molecules could represent a promising strategy for simultaneously modulating protein homeostasis and inflammation. Based on these considerations, in the present study, we investigated the effects of SIX-2G, a [1,2]oxazole-based compound on heat shock proteins (HSPs). Bronchial epithelial cell models, including the CF-derived IB3.1 and 16HBE cell lines, were treated with sub-cytotoxic concentrations of SIX-2G determined by MTT assays, to evaluate its impact on molecular chaperones (Hsp60, Hsp70, and Hsp90) and CFTR at both transcriptional and protein levels performing quantitative real-time PCR, Western blotting, and immunofluorescence analysis. In parallel, the extracellular release of cytokines, chemokines, and matrix remodeling factors was assessed by a multiplex bead-based immunoassay. The results showed that treatment with SIX-2G caused a significant, dose-dependent modulation of Hsp60, Hsp70 and Hsp90 mRNA expression in both cell models, without affecting the total protein levels of these chaperones or CFTR after 24 hours of exposure. On the other hand, immunofluorescence analyses revealed a pronounced redistribution of CFTR-chaperone subcellular colocalization, suggesting that SIX-2G primarily influences the spatial organization of proteins rather than levels of such proteins. These early alterations in proteostasis-related processes were accompanied by a marked anti-inflammatory effect. In both IB3.1 and 16HBE cells, SIX-2G significantly reduced the extracellular release of multiple pro-inflammatory cytokines and remodelling-associated factors, including IL-3, IL-6, IL-8, MMP-2, MMP-10, and CCL-5. Overall, our findings indicate that SIX-2G modulates airway epithelial cell responses through early transcriptional regulation and spatial reorganization of the CFTR-chaperone complexes. This multilayered mechanism highlights the therapeutic potential of targeting chaperone-mediated proteostasis and suggests SIX-2G as a promising modulator of the inflammatory stress typically associated with CF.