Journal: npj Drug Discovery

Article Title: Triple potentiator combinations increase the functional correction of several CFTR gating mutants

doi: 10.1038/s44386-026-00061-6

Figure Lengend Snippet: A Function of R352Q-, S549R-, S549N-, W1282X- (TGA stop-codon in position 1282) and N1303K-CFTR was determined by the halide-sensitive YFP-quenching assay upon treatment with single potentiators (VX-445 - 2 µM, VX-770 - 3 µM, bDMC and ABBV-974 - 10 µM, and apigenin - 50 µM, 37 ˚ C) or their combinations in CFBE41o- cells ( n = 3). The N1303K -CFTR conformational defects were partially rescued with VX-661 (3 µM, 24 hours, 37 ˚ C). CFTR activity was measured after maximal phosphorylation with forskolin and IBMX as described in Methods. Data are means ± SEM of three independent experiments. * P < 0.05, ** P < 0.01 by one-way ANOVA followed by Tukey’s post-hoc test. B Heat map of the combinatorial profiling was established by calculating the dual potentiator effects, determined by the halide-sensitive YFP quenching assay (A, n = 3), in relation to their theoretical additivity. Combinatorial profiles were subsequently used to cluster compounds by average linkage analysis, and the distance was determined by Spearman’s rank correlation.

Article Snippet: CFTR modulators VX-445, VX-770 and ABBV-974 (former GLPG1837), as well as forskolin and CFTR Inh -172, were purchased from MedChemExpress.

Techniques: Activity Assay, Phospho-proteomics

Journal: npj Drug Discovery

Article Title: Triple potentiator combinations increase the functional correction of several CFTR gating mutants

doi: 10.1038/s44386-026-00061-6

Figure Lengend Snippet: A Representative traces (left) of the I sc of G551D-CFTR in gene-edited (ge) 16HBE cells activated with forskolin (Fsk, 20 μM) and potentiated with VX-770 (3 μM) followed by VX-445 (2 μM) and apigenin (50 µM) or in reverse order. At the end of the experiment, CFTR was inhibited with CFTR Inh -172 (20 μM). Measurements were performed in the presence of a basolateral-to-apical chloride gradient in the presence of 100 μM amiloride. Quantification of the I sc (right, n = 3) shows the contribution of Fsk, VX-770, VX-445, and apigenin to the maximal current, depending on the order of potentiator addition, expressed as a percentage of the forskolin-stimulated WT-CFTR current in 16HBE14o- cells. N.s. – not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired two-tailed t-test for the successive addition of potentiators. B Immunoblot (top panel) of parental 16HBE14o- or 16HBEge expressing endogenous WT- or G551D-CFTR, respectively. CFTR was visualized with 660 antibody, and anti–Na + /K + -ATPase antibody served as a loading control. The empty arrowheads indicate the mature, complex glycosylated CFTR (C-band), the filled arrowheads show the immature, core glycosylated protein (B-band). The relative abundance of the C-band was quantified by densitometry (lower panel, n = 4). C Representative traces (left) and quantification of the I sc (right) in HNE isolated from one patient homozygous for G551D-CFTR upon activation with forskolin (Fsk, 20 μM) and potentiation with VX-770 (3 μM), VX-445 (2 μM) and apigenin (50 μM) expressed as percentage of mean Fsk-induced WT-CFTR currents in HNE from 10 healthy donors. Measurements were performed as in panel A. N.s. – not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired two-tailed t-test for the successive addition of potentiators. D Inside-out patch clamp recording of G551D-CFTR in the presence of 2 mM ATP + 300 nM PKA (black), +50 nM Vx-770 (green), +1 µM VX-445 (blue), +1 µM apigenin (blue), and +50 µM P-dATP (orange, ’CCC’ (channel counting cocktail)). Membrane potential was -80 mV, scales of time and amplitude are shown, gray section illustrates channel closure upon simultaneous removal of ATP, PKA and compounds. E Calculated absolute values of P o for the four segments ( n = 7-8) obtained as described in Methods. * P < 0.05, ** P < 0.01 by one-way ANOVA followed by Tukey’s post-hoc test. F ) Effect of VX-770 (3 μM) or apigenin (50 μM) on the potency of VX-445 for the phosphorylated G551D-CFTR potentiation, measured by halide-sensitive YFP quenching assay in CFBE41o- cells ( n = 3). Values are expressed as a percentage of the response to 3 μM VX-770. G Effect of VX-770 (3 μM) or VX-445 (2 μM) on the potency of apigenin for the phosphorylated G551D-CFTR potentiation, determined by the YFP quenching assay in CFBE41o- cells ( n = 3). Data in ( A , B , E – G ) are means ± SEM of the indicated number of independent experiments. Data in ( C ) are means ± SD of three measurements.

Article Snippet: CFTR modulators VX-445, VX-770 and ABBV-974 (former GLPG1837), as well as forskolin and CFTR Inh -172, were purchased from MedChemExpress.

Techniques: Two Tailed Test, Western Blot, Expressing, Control, Isolation, Activation Assay, Patch Clamp, Membrane

Journal: npj Drug Discovery

Article Title: Triple potentiator combinations increase the functional correction of several CFTR gating mutants

doi: 10.1038/s44386-026-00061-6

Figure Lengend Snippet: A Representative traces (left) and quantification (right, n = 3) of the I sc of VX-661-corrected (3 µM, 24 hours, 37˚C) W1282X-CFTR in 16HBEge activated with forskolin (Fsk, 20 μM) and potentiated with VX-770 (3 μM) followed by VX-445 (2 μM) and apigenin (50 µM) or in reverse order. B Quantification ( n = 3) of the I sc of VX-661 + VX-445 corrected W1282X-CFTR in 16HBEge. C Representative traces (left) and quantification of the I sc (right) in 1 µM SMG1i + 3 µM VX-661 + 2 µM VX-445 treated (24 hours, 37˚C) HNE isolated from one patient homozygous for W1282X-CFTR upon activation with forskolin (Fsk, 20 μM) and potentiation with VX-770 (3 μM) and apigenin (50 μM) expressed as percentage of mean Fsk-induced WT-CFTR currents in HNE from 10 healthy donors. D Representative traces (left) and quantification (right, n = 3) of the I sc of VX-661-corrected (3 µM, 24 hours, 37˚C) N1303K-CFTR in 16HBEge activated with forskolin (Fsk, 20 μM) and potentiated with VX-770 (3 μM) followed by VX-445 (2 μM) and apigenin (50 µM) or in reverse order. E Representative traces (left) and quantification of the I sc (right) in 3 µM VX-661 treated (24 hours, 37 °C) HNE isolated from one patient homozygous for N1303K-CFTR upon activation with forskolin (Fsk, 20 μM) and potentiation with VX-770 (3 μM), VX-445 (2 μM) and apigenin (50 μM) expressed as percentage of mean Fsk-induced WT-CFTR currents in HNE from 10 healthy donors. Data in ( A ) and ( C ) are means ± SEM of three independent experiments. Data in ( B ) and ( D ) are means ± SD of three measurements. N.s. – not significant, * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired two-tailed t-test for the successive addition of potentiators.

Article Snippet: CFTR modulators VX-445, VX-770 and ABBV-974 (former GLPG1837), as well as forskolin and CFTR Inh -172, were purchased from MedChemExpress.

Techniques: Isolation, Activation Assay, Two Tailed Test

Journal: npj Drug Discovery

Article Title: Triple potentiator combinations increase the functional correction of several CFTR gating mutants

doi: 10.1038/s44386-026-00061-6

Figure Lengend Snippet: A Chemical sites that were subjected to modification in the medicinal chemistry optimization of ML-014. B, C Primary screen of ML-014 analogs as co-potentiator in combination with either VX-770 or VX-445 for potentiation of G551D- ( B ) or N1303K-CFTR ( C ) expressed in CFBE41o- cells. The relative transport activity of CFTR variants was measured by the halide-sensitive YFP-quenching assay and is expressed as percentage of the triple potentiator combination VX-770 + VX-445+apigenin (3P). Data are means of 4 technical replicates ± SD. D Main Structure-Activity Relationships derived from modification of ML-014. E Structure and activity of the most representative analogs (see text for the description of the compounds).

Article Snippet: CFTR modulators VX-445, VX-770 and ABBV-974 (former GLPG1837), as well as forskolin and CFTR Inh -172, were purchased from MedChemExpress.

Techniques: Modification, Activity Assay, Derivative Assay

Journal: npj Drug Discovery

Article Title: Triple potentiator combinations increase the functional correction of several CFTR gating mutants

doi: 10.1038/s44386-026-00061-6

Figure Lengend Snippet: A Representative trace (left) and quantification (right, n = 3) of the dose-response of ML-014 analogs added as third potentiators (1st 3 µM VX-770, 2nd 2 µM VX-445, in the presence of 20 µM forskolin) to potentiate VX-661 corrected (3 µM, 24 hours, 37˚C) N1303K-CFTR in 16HBEge. Data are means ± SEM of three independent experiments. B Efficacy of triple potentiator combinations for the functional correction of N1303K-CFTR in HNE epithelia. Representative traces (left) and quantification (right, n = 3) of the I sc of VX-661 corrected (3 µM, 24 hours, 37˚C) N1303K-CFTR in homozygous HNE activated with forskolin (Fsk, 20 μM) and potentiated with VX-770 (3 μM) followed by VX-445 (2 μM) and ML-014 analogs (14 µM). Data are means ± SD of three measurements. * P < 0.05, ** P < 0.01 by unpaired two-tailed t-test in comparison to DMSO control. C, D Efficacy of ML-051 and apigenin for the potentiation of S549R-CFTR ( C ) and I507del-CFTR ( D ) in the presence of the Trikafta modulator combination. Representative traces (left) and quantification (right, n = 3–7) of the I sc of 3 µM VX-661 + 2 µM VX-445 corrected (24 hours, 37 °C) S549R-CFTR and I507del-CFTR in CFBE41o- activated with forskolin (Fsk, 20 M) and potentiated with VX-770 (3 μM) followed by apigenin (50 µM) or ML-51 (10 µM). Data are means ± SEM of 3–7 independent experiments.

Article Snippet: CFTR modulators VX-445, VX-770 and ABBV-974 (former GLPG1837), as well as forskolin and CFTR Inh -172, were purchased from MedChemExpress.

Techniques: Functional Assay, Two Tailed Test, Comparison, Control

Journal: Cell Reports Medicine

Article Title: Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modeling

doi: 10.1016/j.xcrm.2026.102692

Figure Lengend Snippet: Conversion of S-diff HNECs into 3D organoids and CFTR function measurements (A) Graphic illustration showing the workflow of generating airway organoids from epithelial fragments of S-diff HNECs, and application in forskolin-induced swelling assays. (B) Representative bright-field images of cystic airway organoids formed after 48 h from S-diff monolayer cultures. Scale bar, 250 μm. (C) Immunofluorescent (top) and bright-field (bottom) images of S-diff HNEC-derived organoids of β-tubulin IV (yellow, left top), MUC5AC (purple, middle top), p63 (red, right top), and DAPI (cyan). Scale bar, 10 μm. (D) Representative images of forskolin-induced swelling determined with calcein green AM ester-stained HC and CF (F508del/F508del) organoids, with images taken before stimulation with forskolin (t = 0, left) and 60 min after stimulation (t = 60 min, right). Scale bar, 500 μm. (E and F) Comparison of FIS between HC and CF airway organoids derived from S-diff HNECs (both n = 3 independent donors) that were unstimulated or stimulated with forskolin (Fsk). (G) CF F508del homozygous organoids derived from S-diff HNECs ( n = 3 independent donors) were pre-treated with vehicle or CFTR correctors, VX-809, or VX-661/VX-445 for 48 h. Afterward, FIS was determined following acute stimulation with Fsk, VX-770, or vehicle. (H) FIS responses in CF F508del/S1251N organoids ( n = 3 donors) following acute stimulation with VX-770. (I) Schematic overview of the comparison between FIS responses in airway organoids derived from ALI- vs. S-diff cultures. (J) Correlation plot of FIS responses between ALI-derived and S-diff-derived organoids from the same CF donors (Pearson r = 0.90, 95% confidence interval, 0.70–0.97; p < 0.0001). (K) FIS in organoids generated from HC basal cells expanded to passage 3 or 7 before S-diff and organoid formation ( n = 2 donors). FIS results are depicted as (E) the percentage change in surface area relative to t = 0 (normalized area) measured at 15-min time intervals for 60 min or (F–H and K) as area under the curve (AUC) plots (t = 60 min). Data are presented as mean ± SD and individual data point. Statistical significance was tested using a (F and I) two-way ANOVA with Dunnett’s multiple comparison test, (G and H) Tukey’s multiple comparison test, (J) Pearson correlation analysis. ns, non-significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: VX-445 , MedChemExpress , Cat#HY-11177.

Techniques: Derivative Assay, Staining, Comparison, Generated