© Copyright 2015 Galapagos NV

Poster available online at:

www.glpg.com

Defects in CFTR that result in Cystic Fibrosis can be broadly categorized into three processes: translational, folding/maturation, and functional. To address the underlying causes affecting the folding/maturation and functional properties of CFTR, two biomolecular activities are required, namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Combined, these activities allow chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance.

Previously, we reported the discovery of new corrector molecules via a HTS screen on CFBe41o- cells harboring HRP-tagged F508del CFTR and on U2OS cells expressing ProLink-tagged F508del CFTR. The corrector compounds exhibited good improvement of channel activity in primary cells derived from F508del CFTR homozygous patients as measured by trans-epithelial clamp circuit (TECC).

Kinetic studies of Compound B in the CFBe41o- cells harboring HRP-tagged F508del CFTR1 demonstrated a difference in the correction rates compared to C18, suggesting that both compounds function by a distinct mechanism(s). Additional kinetic studies using the same compounds and cell system but using Brefeldin to block addition of newly synthesized F508del CFTR at the plasma membrane, showed differences in F508del CFTR levels. Compounds C18 and B prolong the membrane residence time of corrected F580del CFTR. Combination of these compounds further improved stabilization of F508del CFTR at the cell-surface.

Stable cell lines containing AVI-tagged CFTR (WT and F508del) in CFBe41o- cells were generated to look at cell surface expression of CFTR, allowing for following the faith of CFTR in cells.

Insight into the mechanisms of correctors and potentiators

Sara Musch, Corina Balut*, Ann Vandevelde, Tim Vortherms*, Luc Nelles, Chris Tse*, Katja Conrath

Galapagos NV, Generaal De Wittelaan L11A3, 2800 Mechelen, Belgium. * AbbVie, 1 North Waukegan Road, North Chicago, IL 60064, US. # Cellular Protein Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Conclusions Various assays were used for better understanding of

previously identified novel mechanism correctors. Time needed for correction as well as residence time of corrected F508del CFTR are influenced. A novel cellular assay using AVI-tagged F508del CFTR was developed to visualize by in vivo biotinylation the surface rescued F508del CFTR. This assay can be used for quantification of rescued CFTR levels as well as looking into recycling and trafficking of F508del CFTR, influenced by correctors

Studies using F508del/F508del organoids showed functional rescue of F508del CFTR using correctors and GLPG1837 as potentiator

NACFC-2015

Poster #70

CFTR F508del – HRP in CFBe41o-

G. Lukacs

Residence time of HRP-F508del CFTRCorrection of HRP-F508del CFTR in time

AcknowledgementsWe would like to thank Dr. Gergely Lukacs for providing the CFBe41o- cells. We would also thank the Cystic Fibrosis Foundation for providing C18. We would like to thank Anabela Ramalho and Kris De Boeck for providing the F508del/F508del organoids.

References1. Phuan, P-W, et.al. (2014) Synergy-Based Small-Molecule Screen Using a Human Lung Epithelial Cell Line Yields DF508-CFTR

Correctors That Augment VX-809 Maximal Efficacy. Mol Pharmacol 86:42–51.

SummaryIn summary, these studies have elucidated additional understanding of the interplay between

correctors/potentiators and CFTR. We gained more insight on how our corrector molecules do work using several assays to evaluate the impact of corrector/potentiator combinations on the rescue of F508del CFTR, and to understand the contribution of each component in combination cocktails.

E-mail: rd@glpg.com

AVI-tag was inserted in ECL of CFTR allowing for enzymatic conjugation of a single biotin on the tagged target, using the biotin ligase (BirA) from E. coli

Degradation rate can be followed by binding fluorescently-tagged streptavidin at cell surface and following the loss of signal

Western blot, CFTR-F508del-AVI Total protein Immunofluorescence, Cell surface CFTR-F508del-AVI Internalization

Rescue of F508del/F508del CFTR in organoids

Organoids from a F508del/F508del patient were prepared and treated with a combination of two correctors for 24 hours. Organoid swelling was followed in time after stimulation with forskolin and GLPG1837.

Cm

pd

C (

3µM

)

Cmpd C (3µM)

AVI-CFTR F508del in CFBe41o-