Identification of GLPG/ABBV-2737, a Novel Class of Corrector, Which Exerts Functional Synergy With Other CFTR Modulators

Abstract

The deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) causes a severe defect in folding and trafficking of the chloride channel resulting in its absence at the plasma membrane of epithelial cells leading to cystic fibrosis. Progress in the understanding of the disease increased over the past decades and led to the awareness that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit. Today, there remains an unmet need for identification and development of more effective CFTR modulator combinations to improve existing therapies for patients carrying the F508del mutation. Here, we describe the identification of a novel F508del corrector using functional assays. We provide experimental evidence that the clinical candidate GLPG/ABBV-2737 represents a novel class of corrector exerting activity both on its own and in combination with VX809 or GLPG/ABBV-2222.

Keywords: chloride channel; cystic fibrosis; cystic fibrosis transmembrane conductance regulator (CFTR); electrophysiology; protein misfolding.

FIGURE 1

Effect of HIT1 on F508del CFTR trafficking and function. (A) Dose response of 24 h treatment with HIT1 in CSE-HRP assay, % F508del CFTR expression at plasma membrane was normalized using VX809 correction as 100% in (A–D) and each concentration was tested in duplicate. (B) Dose–response of 24 h treatment with HIT1 in combination with 3 μM VX809 in CSE-HRP assay. (C) Dose–response of 24 h treatment with HIT1 in CSE-MEM assay. (D) Dose–response of 24 h treatment with HIT1 in combination with 3 μM VX809 in CSE-MEM assay. (E) CSE-MEM cells were incubated with compound for 24 h. Band C, mature complex-glycosylated F508del CFTR; Band B, immature core-glycosylated CFTR. (F) F508del/F508del HBE cells were incubated with compound for 24 h, CFTR channel was activated with 10 μM forskolin and 3 μM potentiator. ^∗∗∗ denotes a p-value < 0.001 compared to DMSO treatment. (G) Average of raw traces of experiments represented in F (n = 4 for each condition in F and G).

FIGURE 2

Progression of efficacy (X-axis) and potency (EC₅₀, Y-axis) of corrector series on F508del CFTR, in a CSE-HRP assay. Each dot represents a single compound. Control used in assay corresponds to compound 15 (a lead compound derived from GLPG2222 series, Wang et al., 2018). (A) Effect of compounds on their own on F508del CFTR rescue. (B) Effect of compounds when combined with a C1 corrector (compound 15). Black dot represents the original hit HIT1, the green dot represents GLPG2737. (C) Schematic representation of the shifts in potency and efficacy comparing Hit1 with GLPG2737 both with and without co-corrector treatment.

FIGURE 3

Effects of GLPG2737 on F508del CFTR maturation and function. (A) Dose–response of GLPG2737 after 24 h treatment in CSE-HRP assay, % F508del CFTR expression at plasma membrane (PM) was normalized to control compound 15 (derived from GLPG2222 series) in (A–D) and each concentration was tested in duplicate. (B) Dose–response of GLPG2737 in combination with 0.5 μM compound 15 (derived from GLPG2222 series) after 24 h treatment in CSE-HRP assay. (C) Dose–response of GLPG2737 after 24 h treatment in CSE-MEM assay. (D) Dose–response of GLPG2737 in combination with 0.5 μM GLPG2222 after 24 h treatment in CSE-MEM assay. (E) CSE-MEM cells were incubated with compound for 24 h. Cell lysates were loaded on gel and Band B/C was detected using 596 antibody (Band C, mature complex-glycosylated F508del CFTR; Band B, immature core-glycosylated CFTR. (F) F508del/F508del HBE cells were incubated with indicated corrector compound(s) for 24 h, CFTR channel was activated with 10 μM forskolin and 1.5 μM potentiator. ^∗∗∗ denotes a p-value < 0.001 compared to DMSO treatment. ^∘∘∘ denotes a p-value < 0.001 compared to GLPG2222 + GLPG2737 treatment. (G) Average of raw traces of experiments represented in F (n = 3–6 for each condition in F and G) (H) Dose–response of 24 h treatment with GLPG2737 in combination with 0.15 μM GLPG2222 (black) or without (gray) and 1.5 μM GLPG3067 (tested in duplicate for each concentration).

FIGURE 4

Effects of single corrector or combinations of correctors on F508del CFTR channel function as measured by TECC in F508del/F508del HBE cells. (A) Compounds (0.15 μM GLPG2222 and/or 1 μM GLPG2737 and/or 1.5 μM GLPG2451) were incubated on cells for 24 h, CFTR channel was activated with 10 μM forskolin and change in short circuit current was measured in three to six replicates for each condition ^∗∗∗ denotes a p-value < 0.001 compared to DMSO treatment. ^∘∘∘ denotes a p-value < 0.001 compared to GLPG2222 + GLPG2737 + GLPG2451 treatment. (B) Compounds (0.15 μM GLPG2222 and/or 1 μM GLPG2737) were incubated on cells for 24 h, CFTR channel was activated with 10 μM forskolin followed by 1.5 μM potentiator GLPG2451 and change in short circuit current was measured after forskolin activation and after GLPG2451 addition. n = 3 for each condition and p-values are denoted with + meaning p < 0.01 comparing similar chronic conditions from panel (A) with acute potentiator conditions in panel (B).

FIGURE 5

Single-channel behaviors of F508del CFTR expressed in CHO cells pre-treated with corrector(s) GLPG2222, GLPG2737 and or potentiator GLPG3067. (A) Representative single-channel current traces of F508del CFTR in the presence of 0.5 μM GLPG2222 + 10 μM GLPG3067 + 1 μM GLPG2737 (upper trace) and after washout of GLPG2737 (lower trace) from a cell pre-treated with 0.5 μM GLPG2222 + 10 μM GLPG3067 + 1μM GLPG2737. Kinetic parameters (Po, τ_o, and τ_c) are shown on the right. (B) Representative single-channel current traces of F508del CFTR in the presence of 0.5 μM GLPG2222 (upper panel), after addition of 10 μM GLPG3067 (middle panel) and additional application of 1 μM GLPG2737 (lower panel) from a cell pre-treated with 0.5 μM GLPG2222. Kinetic parameters (Po, τ_o, and τ_c) are shown on the right. The statistical analysis of single-channel kinetic parameters was described in “Materials and Methods” Section, every condition was tested at least three times. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.005.

FIGURE 6

Effects of GLPG2737 on normal human bronchial epithelial cells (NHBE) and HEK cells expressing WT CFTR. GLPG2737 dose response effect on WT CFTR activity in HEK293 cells after 24 h stimulation (A) or acute incubation (B) and activation using 100 nM FSK. C. GLPG2737 dose response effect on CFTR activity in NHBE cells after acute addition (0.3 μM forskolin) (tested in duplicate at each concentration for panels A–C). (D) NHBE treated for 24 h with 1 μM GLPG2737 or DMSO (control), CFTR channel was activated with either 0.1 μM or 0.3μM forskolin, measured I_sc shown in gray. After forskolin treatment, potentiator GLPG2451 (10 μM) was added to the GLPG2737 treated cells, further increase in I_sc shown in white bar. n = 2–3 for each condition, p < 0.01 when comparing GLPG2737 with DMSO treated cells after forskolin treatment (all cases) and p > 0.05 when comparing GLPG2737 with DMSO treated cells after forskolin + potentiator treatment (all cases). (E) Representation of the average raw traces represented in panel A (0.3 μM forskolin). Light gray line represents cells without forskolin. Green line represents GLPG2737 treated cells. Black line represents cells treated only with forskolin.

FIGURE 7

YHA assay using CFTR mutant overexpression in HEK293 cells. Cells were incubated for 24 h with either a dose range of GLPG2737, GLPG2222 or VX809. After washing, cells were triggered with forskolin (50 μM for F508del, E92K, V232D and F508del/I539T, 10 μM for P67L or 1 μM for F508del/G550E) and 0.5 μM potentiator and YFP fluorescence was measured. Efficacy was determined using 10 μM GLPG2222 as positive control and DMSO as negative control in the assay. (A) Table with potency and efficacy obtained for the mutants and compounds tested. (B) Dose–response curves of GLPG2737. (C) Dose–response curves of GLPG2222. (D) Dose–response curves of VX-809. For panels (B–D), E92K mutant is represented in black, F508del mutant in blue, F508del/I539T mutant in green and V232D mutant in gray, all concentrations are tested in duplicate.