1. POTENTIATORS: HOW DO THEY IMPACT THE FATE OF CFTR DURING BIOGENESIS? 1 Cellular Protein Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 2 Galapagos NV, Generaal De Wittelaan L11A3, 2800 Mechelen, Belgium; 3 AbbVie, 1 North Waukegan Road, North Chicago, IL 60064, US. Conclusions: Defects in CFTR that result in Cystic Fibrosis can be broadly categorized into three processes: CFTR translation, folding/maturation, and function. To address the underlying causes affecting the folding/maturation and functional properties of CFTR, roughly two biomolecular activities are required: correctors to improve CFTR conformation and increase its levels at the cell surface, and potentiators to improve 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. Galapagos developed a broad set of novel potentiators and identified a very potent compound, GLPG1837, that improved channel activity of temperature-rescued F508del-CFTR as well as G551D-CFTR (Figure 1). In Trans-epithelial Clamp Circuit (TECC) assays on primary patient cells the maximal opening of the G551D-CFTR channel exceeded that of Kalydeco (VX-770) by >200% (Figure 1). Moreover, GLPG1837 did not show the adverse effects VX-770 has on rescued F508del-CFTR after chronic treatment (Figure 2). Pulse chase analysis confirmed that chronic treatment of GLPG1837 did not reduce levels of drug-induced F508del-CFTR correction (Figure 4B). To gain insight into the mechanism of action of correctors and potentiator molecules or combinations thereof, we performed radiolabeling of wild-type or F508del-CFTR expressing cells in combination with limited proteolysis (where) or time-of-addition (when) studies. The adverse effect of VX-770 on wild-type CFTR biosynthesis was reduced when this potentiator was added late posttranslationally, after 30 min chase (Figure 3). Investigating impact of correctors and potentiators on protein trafficking (ER versus Golgi-form), and folding and stability of CFTR domains (limited proteolysis) demonstrated modest correction of F508del-CFTR by the individual compound (Figure 4A). However, the combinatorial approach led to a robust rescue in trafficking and functioning of F508del-CFTR without repairing NBD1 misfolding (Figure 4B). The impact of these results on structural and functional correction of F508del-CFTR are discussed in Figure 5.  Adding VX-770 late posttranslationally (after 30-min chase) reduces adverse effect on CFTR folding and stability  Chronic GLPG1837 treatment does not have negative effects on drug-induced F508del-CFTR correction, both functionally and structurally  Novel potentiator GLPG1837 does rescue F508del/F508del ‘corrected’ & G551D/F508del patient cells Figure 2: To determine whether or not the novel potentiator influenced the drug-corrected folding and stability of F508del-CFTR, channel function after chronic (both corrector and potentiator added 24h prior measurement) or acute (corrector 24h and acute measurement of potentiator) treatment of primary HBE F508del/F508del cells (21 days differentiated) using the Transepithelial Clamp Circuit (TECC) assays was evaluated. Whereas chronic treatment of 2uM VX-770 negatively influenced VX-809 correction (B, red arrow) as reported before (Cholon et al.,2014), chronic treatment GLPG1837 up to 2uM clearly did not (A). To further substantiate these results chronic potentiator treatment effects of HRP-tagged F508del-CFTR in CFBe41o- cells (G. Lukacs cell line) at the cell surface (DR corrector = dose-response corrector) were determined. Chronic treatment with VX-770 reduced cell surface stability of drug-corrected HRP-F508del (Veit et al, 2014), but not with GLPG1837 (C). Figure 3: (A) Schematic representation of the time-of-addition experiments. The grey box indicated drug addition and the white box DMSO control. (B) F508del CFTR was expressed in HEK293T cells for 24h. After 20 minutes starvation, cells were pulse with 35 S-Met/Cys for 15 min followed by a 2 hour chase. CFTR was immunoprecipitated using polyclonal anti-NBD1 (Mr Pink). Corrector Cmpd F was added according scheme in A. (C) Impact of potentiator on WT CFTR folding. 3 µM potentiator GLPG18037 or VX770 were added according scheme in A. Addition of VX-770 after 30 min chase had the least effect on CFTR folding and stability (red box). 1: Robust functional rescue by novel potentiators Figure 1: Potentiators from two different series were screened for functional rescue of temperature- corrected F508del-CFTR (A) or G551D-CFTR (C) in CFBE41o- cells using the YFP-halide assay. Potentiation of the channel by all novel compounds, including GLPG1837 (red), were related to VX-770 (blue). Transepithelial Clamp Circuit (TECC)) assays on primary HBE cells from homozygote F508del (B) or G551D/F508del (D) patients showed that GLPG1837 improved channel function ~two-fold compared to the VX-770 rescue ceiling (see blue arrows). Figure 4: (A) Using pulse chase analysis like in Figure 3 rescue effects of each corrector individually on F508del-CFTR was investigated. Compound was present during starvation, pulse and chase. (B) The combinatorial effects of compounds on F508del-CFTR trafficking (ER vs. Golgi form), and the conformation of individual domains NBD1 and NBD2 was investigated after 0h (left panel) and 2h chase (right panel). Radiolabeled lysate was treated with 25 µg/ml proteinase K for 15 minutes on ice. Protease resistant NBD1 and NBD2 fragment were immunoprecipitated using Mr Pink or 596 antibodies, resp. Both novel correctors (Cmpd F or G) worked additive with VX-809 correction (increasing ER and Golgi form), however without repairing the NBD1 conformation. Note that the increase in NBD2’s protease resistance is the consequence of corrected F508del-CFTR folding and trafficking. More importantly, chronic treatment (18 hour prior pulse chase) with GLPG1837 did not negatively influence the drug-correction of F508del-CFTR (indicated by number sign #). 5: How do potentiators contribute to F508del-CFTR rescue? Figure 5: Individual domain folding and the first domain assembly stages of CFTR happen cotranslationally (Kleizen et al., 2005), after which lengthy posttranslational domain assembly stages are needed to finalize the folding. NBD1 misfolds in F508del-CFTR cotranslationally (Hoelen et al., 2010) and as a consequence fails to assemble its domains posttranslationally. Combining two correctors improved F508del-CFTR folding and trafficking significantly compared to single corrector treatment, but without restoring NBD1 domain folding. Chronic treatment of GLPG1837 did not have adverse effect F508del-CFTR correction compared to VX-770 treatment. Relevant questions to ask: Does VX-770 evoke structural changes in CFTR during and/or directly after synthesis? To what extent does corrected F508del–CFTR resemble fully functional wild-type channel? 2: Chronic versus acute potentiator effects on drug- corrected F508del-CFTR Impact of GLPG1837 in HBE F508del+ VX-809 TECC Impact of VX-770 in HBE F508del+ VX-809 TECC Florence Peters 1, Bertrand Kleizen 1, Martin Andrews 2, Steven van der Plas 2, Chris Tse 3, Katja Conrath 2, Ineke Braakman 1 A G551D/ F508del HBE Max level VX-770 F508del/ F508del HBE G551D CFTR – YHA assayF508del CFTR – YHA assay 3: VX-770 reduced CFTR levels when present during biosynthesis A B C D A B C Impact of co-incubation corrector with potentiator on F508del CFTR at the cell surface B C 4: Potentiator GLPG1837 does not compromise drug-correction of newly synthesized F508del-CFTR A B Funding: Cystic Fibrosis Foundation, The Dutch Cystic Fibrosis Society (NCFS), Agentschap voor Innovatie door Wetenschap en Technologie (IWT). References: Cholon et al., Science trans. Med., 2014; Veit et al., Science trans. Med., 2014; Kleizen et al., Mol Cel, 2005; Hoelen et al., PLoSOne, 2010 Acknowledgement: Tim Jensen (CFFT) for providing 596 monoclonal against NBD2  Combinatorial approach (2 correctors and 1 potentiator) leads to robust rescue of F508del-CFTR without repairing NBD1 folding