CHARACTERIZATION OF NOVEL CFTR POTENTIATORS
Conrath, K.2; Swensen, A.3; Gees, M.2; Verdonck, K.2; Van der Plas, S.2; Andrews, M.2; Hwang, T.1 1. Medical Pharmacology and Physiology, University of Missouri, Columbia, MO, USA; 2. Galapagos, Mechelen, Belgium; 3. AbbVie, Chicago, IL, USA
Using YFP-based high-throughput screening assay, we identified novel compounds that can potentiate cAMP-dependent activity of low tempera-ture-rescued F508del CFTR in CFBe41o- cells. A series of reagents was developed further, with improvement in the potency of compounds down to <10nM for a number of new analogues.These compounds were profiled in additional YFP halide assays using other class III and IV CFTR mutants. These assays were performed by transient transfection of CFTR mutant in HEK293 cells and iodide influx was measured after addition of forskolin and potentiator. All compounds tested were able to open channels from the different mutants in an efficient manner. For several class III mutants, the maximal activity of the mutant channel exceeded that of VX-770-treated channel (which was used as comparator in the assay) with iodide influx up to 250% of the maximum obtained by VX-770. Furthermore, our compounds showed higher poten-cy compared to VX-770 in the same assay. A broad set of compounds was evaluated in parallel on low temperature-rescued F508del CFTR and G551D CFTR and a good correlation between potencies in these two assays was observed.One of the compounds (GLPG1837) was characterized in detail with the patch-clamp technique. In inside-out patches excised from CHO cells transiently expressing WT, G551D or F508del CFTR, applications of the compound resulted in reversible potentiation of the activity of the channels pre-activated with protein kinase A and ATP. For WT CFTR, the Po in the presence of 3 mM GLPG1837 is 0.78 ± 0.03 (n = 9) with open time (to) and closed time (tc) constants of 1479 ± 387 ms and 292 ± 26 ms respectively, a result compatible with those seen with VX-770 (Jih KY, Hwang TC. Proc Natl Acad Sci USA. 2013;110:4404-9). However, a 27.5 ± 3.0 fold increase of macroscopic G551D CFTR currents was observed (~10 fold for VX-770 in Jih and Hwang, 2013). For F508del, the Po was dramatically increased to 0.55 ± 0.05 (n = 9) with to = 3290 ± 819 ms and tc = 2182 ± 516 ms.In summary, further characterization of recently identified novel poten-tiators was performed confirming the increased channel opening observed in primary cells using patch clamp.