Mutations in the second cytoplasmic loop of CFTR suggest distinct mode of action between potentiators VX-770 and glpg1837

Abstract

The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified a novel potentiator GLPG1837 by compound screening on mutant CFTR. YFP-halide efflux assays and single channel measurements showed ∼2.5-fold improvement in channel activity by GLPG1837 compared to VX-770 (ivacaftor/Kalydeco) on G551D CFTR (1, 2). GLPG1837 successfully passed the Phase-2 clinical trials and proved to be the first potentiator after VX-770 to show competitive results on G551D patients. To identify potential differences in the mode of actions of these potentiators we studied their effects on CFTR folding and function. Biochemical radiolabeling experiments showed that mutations in the intracellular loop 2 (ICL2) disrupt domain assembly between TMD1 and NBD2, a late folding event in CFTR, but in most cases do not impair CFTR trafficking towards the cell surface. Protease-susceptibility assays showed that VX-770 improved late TMD1 folding of many ICL2 mutations, but GLPG1837 did not. YFP-halide efflux assays showed that these ICL2 mutants had varying effect on channel function, ranging from wild-type-like to function-defective mutants. GLPG1837 restored function of non-CF gating mutant E267K much better than VX-770. Residue E267 in ICL2 electrostatically interacts with K1060 in ICL4 to promote channel opening (3). This indicates that GLPG1837 is more efficient in compensating for this lost interaction. Altogether, our biochemical and functional data suggests that potentiators VX-770 and GLPG1837 have a different mode of action.