METHODS

Introduction

Conclusions

The novel potentiator GP-2 is highly efficacious towards enhancing CFTR function following translational RT of PTCs, and compares favorably with ivacaftor, even during chronic administration.

The combination approach of a RT drug with potentiators may boost nonsense suppression to therapeutic levels of CFTR function.

The combination of RT agents and potentiators deserves exploration in human subjects with nonsense mutations.

• UAB Cystic Fibrosis Research Center (CFRC)

• Galapagos NV

• Premature termination codons (PTCs) in the CFTR gene result in nonfunctional CFTR protein and are the proximate cause of ~11% of CF causing alleles. Use of aminoglycosides has been shown to induce readthrough (RT) of PTCs, restoring CFTR expression and function.

• CFTR potentiator ivacaftor (VX-770) was successfully used in restoring CFTR function in G551D and other related mutations and has also been shown to augment CFTR function following RT by potentiating rescued CFTR channels. However, ivacaftor may have deleterious effects on CFTR stability, thus the evaluation of alternative potentiators is warranted.

• We hypothesize that novel CFTR potentiators alone and in combination with translational RT could improve CFTR activity and function to confer a significant therapeutic benefit to CF patients with PTCs.

GP-2 activity in primary cells pre-treated with readthrough agents

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Fig. 4. Effect of GP-2 vs. VX-770 in primary human bronchial epithelial cells (ΔF508/G542X). HBE cells were pretreated with RT agents (NB124 or G418) for 48 hrs followed by Isc measurements. (A) FSK+ VX770 & (B) FSK+GP-2 stimulated Isc in NB124 pre-treated cells. (C) GP-2 induced Isc was significantly greater in NB124 pre-treated cells compared to VX-770 (P<0.05) (D) G418 pre-treated HBE cells also exhibit significant increase in CFTR potentiation even at 0.1 µM and 1 µM and GP-2 was been shown to be efficacious potentiator compared to VX-770 * P<0.05, ** P<0.001, **** P<0.0001

Novel Potentiators Augment Efficacy of Translational Readthrough in CFTR Nonsense Mutations

Venkateshwar Mutyam1, Xiaojiao Xue1, David M. Bedwell1, Timor Baasov2, Martin Andrews3, Steven van der Plas3, Katja Conrath3, Steven M. Rowe1

  1CFRC, University of Alabama at Birmingham, Birmingham, AL., 2Technion-Israel Institute of Technology, Haifa, Israel, 3Galapagos NV, Generaal De Wittelaan L11A3, 2800 Mechelen, Belgium.

Cell Culture• Fisher Rat Thyroid (FRT) cells: FRT’s were stably transduced with CFTR G542X, R1162X and W1282X stop

codons, along with matched CFTR WT control cDNA using the Flp-In system (Invitrogen). To study the functional consequences of amino acids (AAs) inserted during PTC suppression, FRT CFTR

missense mutations with AA substitutions at the 542 position (Arg,G542A; Trp, G542W; Cys, G542C) were created.

• Primary cells: Human bronchial epithelial cells derived from lung explants carrying a nonsense alleles G542X in trans with F508del CFTR (G542X/ΔF508) were seeded on to permeable supports, grown in differentiating media for at least 6-8 weeks until terminally differentiated.

Conductance (transepithelial chloride conductance, TECC) Assay• For conductance (Gt) measurements cells were seeded on costar 24 well 0.4 uM permeable supports (~4.5

million cells/well) and maintained at 37°C with 5% CO2. Cells were grown to confluence and treated G418 or NB124 (250 µg/ml) for 48 hrs.

• CFTR activity was measured as a change from baseline conductance following the addition of forskolin (20 µM), potentiators (GP-2, GP-4, VX-770; 10 µM) and then CFTRInh-172 (10 µM).

Ussing chamber• Short circuit current (Isc) in primary cells was measured using this voltage clamped apparatus, where baseline

measurement was taken before the apical addition of 100 µM Amiloride. A chloride gradient was then imposed by replacing the apical ringer’s solution with a low chloride Ringer’s with simultaneous addition of amiloride.

• FSK was administered to stimulate CFTR activity before Ivacaftor (VX-770) followed by the addition of CFTR specific inhibitor, CFTRInh-172.

Methods

Acknowledgements

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GP-2 activity on FRT G542 amino acid variants

Combination approaches: GP-2 activity in FRT G542X cells

Fig. 3. Effect of GP-2 vs. VX-770 on FRT G542 amino acid variants. Representative Gt tracings of FRT monolayers expressing AA variants along with WT, subjected to acute treatment with (A)VX-770 or (B)GP-2. Summary of Gt induced (C)FSK+ VX-770 and (D)FSK+GP-2. The FSK induced CFTR activity was further enhanced by addition of potentiators (VX-770 or GP-2) and was significantly higher in WT, G542W, G542R and G542C compared to FRT (no CFTR) cells. (E) GP-2 was more efficacious as a potentiator compared to VX-770* P<0.05, **** P<0.0001

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Fig.1. Effect of potentiators on FRT cells with stopcodon mutants pretreated with G418 : FRT cell monolayers with PTC constructs (G542X, W1282X, R1162X) were pretreated with G418 for 48 hrs followed by conductance (Gt) measurements with acute addition of FSK, potentiators, and CFTRInh-172 (A) Dose response (ranging from 3 nM to 10,000 nM) of GP-2, VX-770 in FRT G542X cells and (B) FRT W1282X cells. Scatter plots representing maximum efficacy of potentiators at 10 μM in (C) FRT G542X cells and (D) FRT W1282X cells. (E) Enhanced CFTR activity in the presence of 10 μM potentiators in FRT R1162X cells. In all the three PTC mutant cell lines, GP-2 is most efficacious potentiator compared to VX-770. Potentiators had no effect on FRT parental cells without CFTR (data not shown) indicating specificity. **** P<0.0001

Dose response of GP-2 & VX-770 in FRT cells with CFTR PTC mutations following readthrough

Fig. 2. Effect of chronic GP-2 treatment on FRT G542X cells. (A) Representative conductance (Gt) tracings of FRT cell monolayers (n=3) expressing CFTR mutant G542X pretreated with G418 in combination with potentiators ( VX-770 or GP-2) or correctors (VX-809) for 48 hrs (B) FSK levels of different treatment combinations. G418 induced readthtrough CFTR activity is significantly enhanced in the presence of VX-809 and VX-770 or GP-2 as compared to VX-809 or VX-770 or GP-2 alone. In this combination therapy, GP-2 induced potentiation is comparatively higher than VX-770.

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