Molecular Mechanism of Action of Trimethylangelicin Derivatives as CFTR Modulators

Abstract

The psoralen-related compound, 4,6,4′-trimethylangelicin (TMA) potentiates the cAMP/PKA-dependent activation of WT-CFTR and rescues F508del-CFTR-dependent chloride secretion in both primary and secondary airway cells homozygous for the F508del mutation. We recently demonstrated that TMA, like lumacaftor (VX-809), stabilizes the first membrane-spanning domain (MSD1) and enhances the interface between NBD1 and ICL4 (MSD2). TMA also demonstrated anti-inflammatory properties, via reduction of IL-8 expression, thus making TMA a promising agent for treatment of cystic fibrosis. Unfortunately, TMA was also found to display potential phototoxicity and mutagenicity, despite the fact that photo-reactivity is absent when the compound is not directly irradiated with UVA light. Due to concerns about these toxic effects, new TMA analogs, characterized by identical or better activity profiles and minimized or reduced side effects, were synthesized by modifying specific structural features on the TMA scaffold, thus generating compounds with no mutagenicity and phototoxicity. Among these compounds, we found TMA analogs which maintained the potentiation activity of CFTR in FRT-YFP-G551D cells. Nanomolar concentrations of these analogs significantly rescued F508del CFTR-dependent chloride efflux in FRT-YFP-F508del, HEK-293 and CF bronchial epithelial cells. We then investigated the ability of TMA analogs to enhance the stable expression of varying CFTR truncation mutants in HEK-293 cells, with the aim of studying the mechanism of their corrector activity. Not surprisingly, MSD1 was the smallest domain stabilized by TMA analogs, as previously observed for TMA. Moreover, we found that TMA analogs were not effective on F508del-CFTR protein which was already stabilized by a second-site mutation at the NBD1-ICL4 interface. Altogether, our findings demonstrate that these TMA analogs mediate correction by modifying MSD1 and indirectly stabilizing the interface between NBD1 and CL4.

ORKAMBI-Mediated Rescue of Mucociliary Clearance in Cystic Fibrosis Primary Respiratory Cultures Is Enhanced by Arginine Uptake, Arginase Inhibition, and Promotion of Nitric Oxide Signaling to the Cystic Fibrosis Transmembrane Conductance Regulator Channel

“…One hour before the experiments, the cells were treated with 10 mM CB-1158 drug or DMSO control, as required. Cells were then lysed in modified radioimmunoprecipitation assay buffer [50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4, 0.2% (v/v) SDS, and 0.1% (v/v) Triton X-100] containing a protease inhibitor cocktail (Roche) for 10 minutes, and the soluble fractions were analyzed by SDS-PAGE on 6% gels, as described previously (Laselva et al, 2018a;Molinski et al, 2018). After electrophoresis, proteins were transferred to nitrocellulose membranes and incubated in 5% (w/v) milk, and CFTR bands were detected using the human CFTR-NBD2-specific muring mAb 596 (1:500; University of North Carolina Chapel Hill).…”

Activity of lumacaftor is not conserved in zebrafish Cftr bearing the major cystic fibrosis‐causing mutation

“…After 18 hours, the cells expressing F508del‐hCFTR‐GFP or F507del‐zCftr‐GFP were treated with 3 μM VX‐809 or DMSO for 24 hours at 37 or 27°C. The cells were then washed with PBS and the blue membrane potential dye (dissolved in chloride‐free buffer as described,) which can detect changes in TM potential, was added to the cells for 40 minutes at 37 or 27°C. Changes in fluorescence were detected using a fluorescence microplate reader (SpectraMax i3; Molecular Devices) at 37 or 27°C.…”

Anti-Infectives Restore ORKAMBI® Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa

“…CFTR was stimulated with 10 µM Forskolin (Sigma-Aldrich, St. Louis, MO, USA) and 1 µM VX-770 (Selleck Chemicals) for cells treated with VX-809 for 24 h. The assay was terminated with 10 µM CFTRinh172 (Cystic Fibrosis Foundation Therapeutics). The changes in membrane potential were normalized to the point before addition of agonist and to the DMSO control response [22,35].…”

“…P. aeruginosa (lab strain PAO1) exposure has been shown to reduce corrector-mediated rescue (VX-809 or VRT-325) of CFTR in human bronchial epithelial cells (HBE) [16][17][18] and stimulate the expression of the pro-inflammatory cytokines, IL-6 and IL-8 [18,19]. On the other hand, 4,6,4 -trimethylangelicin (TMA), which is a dual-acting compound (CFTR corrector and potentiator), has been shown to exert its action by interacting directly with the CFTR protein, and reducing P. aeruginosa-dependent IL-8 secretion [20][21][22][23][24].…”