Characterization of the Mechanism of Action of RDR01752, a Novel Correc‐
tor of F508del-CFTR
Miquéias Lopes-Pacheco, Iris A.L. Silva, Mark J. Turner, Graeme W. Carlile,
Elvira Sondo, David Y. Thomas, Nicoletta Pedemonte, John W. Hanrahan,
Margarida D. Amaral
PII: S0006-2952(20)30369-5
DOI: https://doi.org/10.1016/j.bcp.2020.114133
Reference: BCP 114133
To appear in: Biochemical Pharmacology
Received Date: 22 April 2020
Revised Date: 30 June 2020
Accepted Date: 30 June 2020
Please cite this article as: M. Lopes-Pacheco, I.A.L. Silva, M.J. Turner, G.W. Carlile, E. Sondo, D.Y. Thomas, N.
Pedemonte, J.W. Hanrahan, M.D. Amaral, Characterization of the Mechanism of Action of RDR01752, a Novel
Corrector of F508del-CFTR, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp.2020.114133
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1 Characterization of the Mechanism of Action of RDR01752, a Novel
2 Corrector of F508del-CFTR
3 Running title: Rescue of F508del-CFTR by corrector RDR01752
5 Miquéias Lopes-Pacheco1
, Iris A. L. Silva1
, Mark J. Turner2,4, Graeme W. Carlile3,4
6 Elvira Sondo5
, David Y. Thomas3,4, Nicoletta Pedemonte5
, John W. Hanrahan2,4
7 Margarida D. Amaral1
1University of Lisboa, Faculty of Sciences, BioISI – Biosystems & Integrative Sciences
9 Institute, Lisboa, Portugal
10 2Department of Physiology, McGill University, Montréal, Québec Canada
11 3Department of Biochemistry, McGill University, Montréal, Québec Canada
12 4CF Translational Research Centre, McGill University, Montréal, Québec Canada
13 5UOC, Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy
14
15
16 Counts:
17 Abstract: 238
18 Main text: 3845
19 References: 40
20 Main Figures: 7
21
22
2
23 Acknowledgements
24 Work funded by UIDB/04046/2020 and UIDP/04046/2020 center grants (to BioISI) from
25 FCT, Portugal and research grant ERARE15-pp-010/JTC 2015 INSTINCT from FCT,
26 Portugal (to MDA) and from CF Canada, FRQS and CIHR (to JWH and DYT). The
27 authors thank Sofia Correia and Luís Marques (both from BioISI) for technical support
28 and Cystic Fibrosis Foundation Therapeutics (CFFT, USA) for C18 compound and
31 Conflict of interest statement
32 There is no conflict of interest related to this work.
33
34 ABSTRACT
35 Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most
36 prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this
37 mutation still face several disease-related symptoms. Thus, more potent compound
38 combinations are still needed. Here, we investigated the mechanism of action (MoA) of
39 RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by
40 RDR01752 was assessed by biochemical, immunofluorescence microscopy and
41 functional assays in cell lines and in intestinal organoids. To determine the MoA of
42 RDR01752, we assessed its additive effects to those of genetic revertants of F508del-
43 CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature.
44 Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma
45 membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752
46 produced lower functional rescue. However, in functional assays using intestinal
47 organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy.
3
48 RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W,
49 as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-
50 treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and
51 function compared to each corrector alone. The lack of additivity of RDR01752 with the
52 genetic revertant R1070W suggests that this compound has the same effect as the
53 insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in
54 F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors
55 mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of
56 F508del-CFTR.
57
58 Keywords: cystic fibrosis; protein trafficking; revertants; low temperature; intestinal
59 organoids; drug discovery.
4
60 1. INTRODUCTION
61 Cystic fibrosis (CF) is the most common life-threating autosomal recessive disease
62 among Caucasians, affecting almost 50,000 individuals in Europe [1]. It is caused by
63 mutations in the gene encoding the CF transmembrane conductance regulator (CFTR)
64 protein, which functions as a chloride (Cl-
) and bicarbonate (HCO3
-
) channel at the apical
65 plasma membrane (PM) of epithelial cells. CF-causing mutations cause channel
66 dysfunction, leading to abnormal ion transport and dehydration of epithelia in several
67 tissues [2,3]. Although CF is a multi-organ disease, the respiratory disorder represents the
68 major cause of morbidity and mortality of individuals with CF due to airway obstruction
69 by a thick mucus, chronic inflammation and persistent infections, which ultimately result
70 in respiratory failure [2,3].
71 CFTR protein is composed of two transmembrane domains (TMD1/2), two
72 nucleotide-binding domains (NBD1/2) and a regulatory domain (RD). The TMDs form
73 the pore through which anions are conducted along their electrochemical gradient, while
74 the NBDs regulate channel gating by binding and hydrolyzing ATP and after RD
75 phosphorylation at multiple sites. Interdomain interactions are critical for this complex
76 protein to achieve its native conformation state [4].
77 Over 2,000 CFTR gene variants have been reported so far
78 (http://www.genet.sickkids.on.ca/), with deletion of a phenylalanine at position 508
79 (F508del in NBD1) being the most prevalent and occurring in ~80% of individuals with
80 CF in Europe, albeit with some geographic variability [1]. F508del causes CFTR protein
81 misfolding that is recognized by the endoplasmic reticulum (ER) quality control (ERQC)
82 machinery and prematurely degraded by the proteasome [5]. Rescue of F508del-CFTR
83 was first demonstrated by low temperature incubation of cells heterologously expressing
84 this mutant [6], thus proving that this mutant is both temperature sensitive and rescuable.
5
85 Over the past decade, significant efforts have been put into high-throughput
86 screening (HTS) of small molecule libraries to identify compounds that rescue the
87 F508del-CFTR protein to the PM. To date, there are three correctors approved for clinical
88 use by the Food and Drug Administration (FDA), being two also approved by the
89 European Medicine Agency (EMA) (all combined with potentiator VX-770/ivacaftor):
90 VX-809/lumacaftor, VX-661/tezacaftor and VX-445/elexacaftor (only FDA-approved).
91 In clinical trials, individuals who were F508del-homozygous and treated with either VX-
92 809 or VX-661 plus VX-770 demonstrated a significant, albeit modest, improvement in
93 lung function [7,8]. More recently, VX-445 was added to the co-treatment with VX-
94 661/VX-770 and this triple combination demonstrated greater therapeutic benefit in phase
95 3 clinical trials [9,10], thus leading to its FDA-approval in individuals with CF, aged ≥12
96 years and with the F508del mutation in at least one allele.
97 Despite such progress, individuals with CF still face several disease-related
98 symptoms and complications, including a progressive deterioration of lung function, and
99 thus novel correctors are still needed to achieve more potent combinations. Furthermore,
100 there are other CFTR trafficking mutants that are not efficiently rescued by available
101 correctors, including G85E and N1303K [11,12]. Along these lines, the novel RDR01752
102 compound was identified as a F508del-CFTR traffic corrector in a small-scale screen [13]
103 and demonstrated to thermally stabilize purified murine F508del-NBD1 in vitro [14].
104 However, its mechanism of action (MoA) remains to be elucidated.
105 Here, we investigated the MoA of RDR01752 in cell lines stably expressing either
106 F508del-CFTR or other CFTR mutants, and in F508del/F508del intestinal organoids. The
107 MoA of RDR01752 was explored by analyzing its additive effects to those of previously
108 described CFTR genetic revertants. These are second-site mutations, i.e., in cis with
109 F508del that partially rescue F508del-CFTR. One of these revertants results from removal
6
110 of the arginine-framed motifs (AFT) acting as retention signals (4RK), thus allowing the
111 mutant protein to escape the ERQC [15-17]. Two others work by correcting folding at
112 critical structural pockets present in the 3D-structure of F508del-CFTR that are absent in
113 wild-type (WT)-CFTR. These include G550E that acts by stabilizing the NBD1:NBD2
114 dimer interface [16] and R1070W that restores the NBD1:ICL4 interaction [17-19]. We
115 also investigated the effects of RDR01752 on the DD/AA variant on the background of
116 WT-CFTR, which lacks the double diacidic code necessary for Sec24-CFTR association
117 and ER exit and thus is retained in the ER, although not misfolded [17,20]. Finally, we
118 tested RDR01752 in combination with low temperature and the FDA-approved corrector
119 drugs VX-809/lumacaftor and VX-661/tezacaftor (and compound C18) with and without
120 chronic exposure of the potentiator VX-770/ivacaftor.
7
121 2. MATERIALS AND METHODS
122 2.1. Cell culture. The CF bronchial epithelial (CFBE) cell line stably expressing F508del-
123 CFTR was cultured as before [21]. CFBE cells stably expressing mCherry-Flag-CFTR
124 (WT, F508del, DD/AA variants or carrying G550E, R1070W, 4RK in cis with F508del)
125 were cultured and CFTR expression was induced with doxycycline (Dox; Sigma, MO,
126 USA) 1 µg/mL as described [22]. Fischer rat thyroid (FRT) epithelial cells stably
127 expressing CFTR variants (WT, G85E, R334W, T338I, R347P, F508del, V520F, S549F,
128 G551D, M1101K, N1303K) were cultured as before [23]. All cell lines were maintained
129 in a humidified incubator at 5% CO2 and 37°C, except during low temperature
130 experiments, in which cells were incubated at 27°C for 24h.
131
132 2.2. Chemicals. All reagents were of the highest purity available. Corrector compounds
133 were either commercially obtained: RDR01752 (STK001879, Vitas-M Lab., IL, USA),
134 VX-809, VX-661 and VX-770 (S1565, S7058 and S1144, Selleckchem, TX, USA) or
135 from CFFT: C18. Correctors were diluted in dimethyl sulfoxide (DMSO) and added to
136 cells diluted in 1% FBS supplemented antibiotic-free medium at indicated concentrations:
137 1 to 20µM RDR01752, 1 or 3.7µM VX-809, 5µM VX-661, 5µM C18, 3µM VX-770.
138 Other reagents (all from Sigma, MO, USA, in DMSO solutions) were (final
139 concentrations, unless otherwise stated): 2µM forskolin (Fsk), 50µM genistein (Gen),
140 100µM 3-Isobutyl-1-methylxanthine (IBMX) and 30µM CFTR channel inhibitor
141 CFTRInh-172 (Inh172).
142
143 2.3. Western blotting (WB). Whole-cell lysates were subjected to SDS-PAGE 7% (w/v) gel
144 analysis followed by CFTR detection using monoclonal anti-human CFTR antibody (596
145 [1:3000] from CFFT) as previously [17]. Anti-α-tubulin antibody (1:10,000, Sigma, MO,
8
146 USA) was used as a loading control. CFTR quantification was as described [21] and its
147 processing obtained by the ratio between the mature form and total CFTR (mature and
148 immature forms) and normalized by the loading control.
149
150 2.4. Immunostaining and CFTR Trafficking assay. Twenty-four hours after seeding stably
151 expressing mCherry-Flag-CFTR CFBE cells (carrying wt-, F508del-, DD/AA- or G550E-
152 F508del, R1070W-F508del, 4RK-F508del-CFTR) onto 384-well plates, compounds
153 were concomitantly administered with Dox. After 48h, cells were immunostained and
154 fixed in a protocol without cell permeabilization as described [22]. The mCherry tag
155 allows to quantify the total amount of CFTR protein expressed by each individual cell,
156 while the Flag tag allows for quantification of CFTR exclusively at the PM. After cell
157 imaging, automatic image analysis was performed using a pipeline developed to measure
158 CFTR traffic efficiency [22]. CFTR PM expression was calculated using the deviation
159 score formula as follows: Deviation Score = (CFTR PMcompound – CFTR PMDMSO)/(2 ×
160 SDDMSO). CFTR PM corresponds to the average for all images treated under the same
161 conditions which passed the quality control implemented in the pipeline [22]. The
162 SDDMSO corresponds to the standard deviation for the control condition. All conditions
163 were performed in triplicated in each plate and repeated in at least three independent
164 experiments.
165
166 2.5. HS-YFP assay on the plate reader. Measurements of CFTR activity were carried out
167 on CFBE cells expressing both F508del-CFTR and HS-YFP (YFP-H148Q/I152L) as
168 described [24]. The assay consists of a continuous 14-s fluorescence read-out 2s before and
169 12s after injection of an iodide-containing solution (PBS with I-
at final concentration
170 100mM). Fluorescence quenching rate of I-
influx was determined for the final 11s of the
9
171 data for each well and was fitted to an exponential function to extrapolate initial slope [24].
172 All conditions were performed in triplicated in each plate and repeated in at least three
173 independent experiments.
174
175 2.6. FLIPR membrane potential (FMP) assay. Measurements of FMP to assess CFTR
176 function were performed in FRT cells expressing various CFTR mutants as described [25].
177
178 2.7. Micro-Ussing chamber measurements. Transepithelial electrical resistance (TEER)
179 of CFBE cells growing on Snap-well inserts (Corning-Costar, MA, USA) was measured
180 as before [26]. Briefly, monolayers with resistance ≥450Ωcm2
were mounted in micro-
181 Ussing chambers with perfusion for recordings under open-circuit conditions as described
182 [26]. Changes in transepithelial voltage (Vte) were continuously recorded and equivalent
183 Fsk/IBMX-stimulated short-circuit currents (Ieq–sc) were calculated by Ohm’s law from
184 Vte and Rte (Ieq–sc=Vte/Rte). For FRT cell lines, transepithelial voltage was measured at
185 37°C with continuous stirring by gassing with 95% O2 and 5% CO2 as described [23].
186
187 2.8. Organoid swelling assay. The forskolin-induced swelling (FIS) assay was performed
188 as described [11]. Twenty-four hours after seeding, organoids were stimulated with Fsk
189 with or without a potentiator (VX-770 or genistein) and live-cell imaging was performed
190 using bright field microscopy (Leica DMI6000B) with a 5× objective for 60 min at 37°C.
191 For quantification of the area under the curve (AUC; t = 60 min, baseline = 100%) a
192 CellProfiler-based algorithm was used (Hagemeijer et al., in preparation). Experiments
193 were performed in triplicate and repeated 3-4 times.
194
10
195 2.9. Statistical analyses. Statistical comparisons were made using GraphPad Prism
196 software v.6.01 (GraphPad, CA, USA) and statistical test used for each experiment has
197 been provided in figure legends. Data are presented as mean ± SD of at least three
198 independent experiments. P values <0.05 were considered significant.
11
199 3. RESULTS
200 3.1. RDR01752 rescues F508del-CFTR processing, PM traffic and channel function
201 Incubation of CFBE cells expressing F508del-CFTR with the RDR01752 compound
202 rescued F508del-CFTR processing, resulting in the appearance of the fully-glycosylated
203 form of CFTR (~180 kDa, band C) in a dose-dependent manner with the maximal
204 correction achieved at 10 µM (Fig. 1A,B). This effect was comparable to that obtained
205 for VX-809 or VX-661, and in contrast to that obtained for DMSO (vehicle), which only
206 led to the appearance of the core-glycosylated form of CFTR (~140 kDa, band B). This
207 result was also confirmed by the immunofluorescence detection of the Flag-tag of
208 mCherry-Flag-F508del-CFTR expressed in CFBE cells without cell permeabilization,
209 only in cells treated with RDR01752 or VX-809, but not DMSO (Fig. 1C,D).
210 To assess the ability of RDR01752 to restore F508del-CFTR function, we first measured
211 the rate of HS-YFP quenching induced by iodide influx into cells in CFBE cells stably
212 co-expressing F508del-CFTR and the HS-YFP (Fig. 2A,B). Both RDR01752 (10 µM)
213 and VX-809 demonstrated rescue of F508del-CFTR function, although the efficacy of
214 RDR01752 was lower than that of VX-809. In order to confirm these findings, we then
215 investigated rescue of F508del-CFTR function by RDR01752 in polarized CFBE cells in
216 the Ussing chamber (Fig. 2C-F). A significant increase in equivalent short-circuit current
217 (ΔIsceq) was observed upon Fsk/IBMX stimulation in cells incubated with either
218 RDR01752 or VX-809 and stimulated with potentiator genistein (Gen) versus those
219 incubated with DMSO alone. These data are consistent with the rate of HS-YFP
220 quenching.
221
222 3.2. F508del/F508del intestinal organoids respond positively to RDR01752
12
223 Next, we tested the effects of RDR01752 using the FIS assay in intestinal organoids with
224 the F508del/F508del genotype. Organoids were incubated with RDR01752, VX-809 or
225 VX-661 for 24h and then acutely stimulated (30 min) with Fsk with or without a
226 potentiator (VX-770 or Gen) to further enhance CFTR function (Figs. 3). Significant
227 swelling was observed in organoids incubated with RDR01752, VX-809 or VX-661 and
228 acutely stimulated with either VX-770 or Gen, in contrast to absence of swelling in
229 organoids without any potentiator or DMSO control. Interestingly, similar swelling
230 values were observed for organoids incubated with any corrector plus potentiator
231 combination.
232
233 3.3. RDR01752 increases the rescue of F508del-CFTR PM in cells expressing in cis the
234 genetic revertants G550E and 4RK or low temperature, but not in R1070W or DD/AA
235 variant
236 In order to characterize the mechanism of action (MoA) by which RDR01752 rescues
237 F508del-CFTR, we investigated revertants of this mutant. To this end, CFBE cell lines
238 stably expressing double tagged-F508del-CFTR in cis with the following genetic
239 revertants: G550E, R1070W, or 4RK were incubated with this compound. In parallel
240 CFBE cells expressing WT-CFTR or the traffic-null variant DD/AA (on a WT
241 background) were also treated with RDR01752. Cells were incubated both at 37ºC and
242 low temperature (27ºC), and PM expression of each CFTR variant was assessed by
243 immunofluorescence (Fig. 4A,B, respectively) and normalized to cells incubated with
244 DMSO at 37°C (upper heatmap, first row).
245 Each of the correctors tested RDR01752, VX-809 or VX-661 rescued F508del-
246 CFTR to the PM with similar efficacy (Fig. 4A) which was further enhanced when
247 incubated at 27°C (Fig. 4B). These compounds also increased WT-CFTR PM expression,
13
248 although VX-809 and VX-661 showed higher efficacy than RDR01752. A small additive
249 effect was found for PM levels of WT-CFTR with each of these compounds and low
250 temperature.
251 Analysis of the effects on the revertants demonstrated that RDR01752, similarly
252 to VX-809 and VX-661, is additive to G550E and 4RK in F508del-CFTR PM rescue
253 (Fig.4A), and further additive to that of low temperature (Fig. 4B). In contrast,
254 RDR01752, also similarly to VX-809 and VX-661, did not demonstrate additivity with
255 R1070W (Fig. 4A), thus suggesting that, like VX-809 [17], it might share a common
256 mechanism. Furthermore, an increase in PM expression of DD/AA-CFTR variant was not
257 observed (Fig. 4A), except when cells were incubated at low temperature (Fig. 4B), as
258 before [17], with no significant additive effect elicited by any of the correctors.
259
260 3.4. Rescuing of F508del-CFTR traffic by RDR01752 is not additive to VX-809 or VX-
261 661
262 Next, we tested whether rescuing by RDR01752 is additive to that of other correctors,
263 namely FDA-approved drugs VX-809 and VX-661 or C18 (Fig. 5). We also evaluated
264 whether chronic exposure to VX-770 affects the rescue of F508del-CFTR by RDR01752
265 (Fig. 5). CFBE cells stably expressing mCherry-Flag-F508del-CFTR were incubated with
266 each corrector for 48h and CFTR PM levels were quantified as above. None of the two-
267 corrector combinations further enhance F508del-CFTR PM expression compared to each
268 corrector alone (Fig. 5). Similar to VX-809 or VX-661, rescue of F508del-CFTR by
269 RDR01752 under chronic exposure to a relatively high concentration of VX-770 resulted
270 in a decrease of F508del-CFTR PM expression (Fig. 5). The inhibitory effect of chronic
271 exposure to VX-770 on F508del-CFTR rescue by VX-809 or VX-661 exposure observed
14
272 here, as previously reported by others [27,28], was even more pronounced for any
273 combination of two correctors compared to individual ones.
274
275 3.5. Functional rescue of other CFTR mutants by RDR01752
276 To evaluate the ability of RDR01752 to rescue other CFTR mutants, we used an FMP
277 assay in FRT cells to measure the depolarization that occurs when CFTR PM channels
278 are activated. The nine missense mutants studied are located across the different CFTR
279 domains, namely in: TMD1 (G85E, R334W, T338I and R347P); NBD1 (V520F, S549F
280 and G551D); TMD2 (M1101K); and NBD2 (N1303K). Cells expressing these CF-
281 causing mutations were incubated with either RDR01752 or VX-809 for 24h (Fig. 6). An
282 increase in the Fsk+Gen response was observed in F508del-expressing FRT cells treated
283 with either RDR01752 or VX-809, indicating that CFTR function was rescued by
284 RDR01752, consistent with both the ΔIsceq data and the rate of HS-YFP quenching
285 observed in CFBE cells. Both RDR010752 and VX-809 also rescue CFTR function in
286 R334W-, V520F- and M1101K-expressing cells. However, in this assay only RDR01752
287 (and not VX-809) demonstrated an effect on G85E- and T338I-expressing cells, while
288 R347P-, S549F- and N1303K-expressing cells only responded to VX-809 treatment,
289 albeit the latter at very low levels (Fig. 6). Unexpectedly, no significant differences were
290 observed in CFTR function between G551D- and WT-expressing cells after treatment
291 with either RDR01752 or VX-809 compared to control.
292 To confirm these findings, we investigated the effects of RDR01752 and VX-809, alone
293 or combined, in polarized FRT cells expressing the mutants that showed rescue, namely
294 F508del-, G85E-, N1303K- or R334W-CFTR by Ussing chamber measurements (Fig. 7).
295 An increase of CFTR-dependent Cl–
secretion was observed in F508del-expressing cells
296 incubated with either VX-809 or RDR01752, albeit much lower for the latter. Incubation
15
297 with both compounds combined did not result in greater rescue of F508del-CFTR
298 function in comparison to VX-809 alone. In contrast to F508del, a substantial cAMP
299 response was observed in Ussing chamber measurements for cells expressing R334W-
300 CFTR and treated with only DMSO, which indicates a residual CFTR function. The
301 rescue of R334W-CFTR by RDR01752 was not higher than that by VX-809 (as in the
302 FMP assay) and co-administration of the two compounds increased slightly but not
303 significantly CFTR-dependent Cl–
secretion. Although the rescue of R334W-CFTR by
304 RDR1752 was significantly higher than by DMSO alone in the FMP assay the observed
305 increase in the Ussing chamber measurements was just a trend and not significant.
306 Furthermore, no significant effects were found in G85E- and N1303K-expressing cells in
307 CFTR function after incubation with either RDR01752 or VX-809, alone or combined.
308
309 4. DISCUSSION
310 The aim of this study was to characterize the effects of corrector RDR01752 both in cell
311 lines that stably express F508del-CFTR or other rare CFTR mutations and in intestinal
312 organoids that are F508del/F508del. Furthermore, we investigated the MoA of
313 RDR01752 by evaluating its additivity with available CFTR corrector drugs, genetic
314 revertants of F508del-CFTR, and low temperature.
315 Most CF drug development programs for CFTR modulators have focused on the
316 rescue of F508del-CFTR as the most prevalent disease-causing mutant. Despite
317 significant progress in restoring F508del-CFTR trafficking by corrector drugs, treatment
318 of F508del-homozygous individuals with single correctors (VX-809 or VX-661) in
319 combination with the potentiator VX-770 achieved only modest clinical improvements
320 [7,8]. Significantly greater therapeutic response was recently achieved by adding a second
321 corrector (VX-445) to the previous combination (VX-661/VX-770) [9,10], indicating that
16
322 combination of correctors acting by distinct mechanisms is needed for efficient correction
323 of F508del-CFTR and for clinical benefit of individuals with CF carrying this mutation.
324 Although the MoA of VX-445 is still unknown, its additivity to VX-661 on F508del-
325 CFTR rescue suggests that these compounds act by different modes.
326 Here, we looked into the MoA of the recently described F508del-CFTR corrector
327 RDR01752 [13]. Our biochemical and immunofluorescence data demonstrated that
328 RDR01752 rescues F508del-CFTR processing and PM expression in CFBE cells to levels
329 similar to those of VX-809. However, in previous studies using baby hamster kidney
330 (BHK) cells stably expressing F508del-CFTR, RDR01752 appeared to be less efficacious
331 than VX-809 [13,14]. Our functional data here, in both CFBE and FRT cell lines, also
332 show that RDR01752 is less effective than VX-809 in restoring F508del-CFTR-mediated
333 Cl-
secretion, consistent with previous findings in human bronchial epithelial (HBE) cells
334 (F508del/F508del) [25]. Notwithstanding, as the validation of results in patient-derived
335 specimens is an important step to provide a better prediction of the in vivo efficacy
336 [29,30], we tested here this compound in intestinal organoids (F508del/F508del). Our
337 data revealed that RDR01752 and VX-809/VX-661 can rescue F508del-CFTR function
338 to similar levels in this assay. The higher efficacy observed in the organoids may derive
339 from the fact that different cell systems were used and CFTR processing and function are
340 influenced by the cell background and polarization state [31-33].
341 To investigate the MoA of RDR01752 we analyzed its additivity to revertants that
342 rescue F508del-CFTR by different mechanisms. RDR01752 effects were additive to
343 G550E and 4RK, but not to R1070W as determined by rescue of F508del-CFTR protein
344 to the PM. G550E and R1070W were proposed to act at two distinct CFTR interdomain
345 contact points that are disrupted by F508del [17]: while G550E likely restores the
346 NBD1:NBD2 dimerization interface [16], R1070W restores the NBD1:ICL4 interaction
17
347 [18,19]. Thus, RDR01752, like VX-809, might act similarly to R1070W, i.e., by restoring
348 the anchoring of ICL4 to the NBD1 surface by filling a pocket generated by the absence
349 of F508, as the lack of additivity of these two compounds indicates. Indeed, the
350 replacement of an arginine with a tryptophan at position 1070 (R1070W) helps restore
351 interactions among the aromatic residue that were impaired by the lack of F508del at the
352 NBD1 surface [17-19]. RDR01752, like VX-809/VX-661, has a fused aromatic ring in
353 its structure that can fit into this pocket left empty by F508del [17,34]. This terminal
354 aromatic ring in RDR01752 was also found to be critical for the stabilization of isolated
355 F508del-NBD1 with an additional stabilization effect in presence of ATP, thus suggesting
356 that RDR01752 does not bind to the ATP binding site in NBD1 [14]. This is also in
357 agreement with the observed additivity of RDR01752 to G550E.
358 On the other hand, RDR01752 was also additive to 4RK. This variant (where four
359 arginines in the AFT are simultaneously replaced to lysines) enables some F508del-CFTR
360 protein to traffic to the PM by escaping the ERQC [15,16]. Notably, although RDR01752,
361 VX-809 and VX-661 rescued F508del-4RK-CFTR PM expression, their efficacy was
362 distinct, with the additivity of RDR01752 to 4RK being higher than that of VX-809/VX-
363 661. In turn, the latter correctors were more effective than RDR01752 in rescuing G550E-
364 F508del-CFTR. Altogether, these data suggest that combinations of RDR01752 with
365 compounds that mimic the correction induced by the G550E and 4RK revertants could
366 maximize the rescue of F508del-CFTR.
367 Regarding the additivity to low temperature, RDR01752, VX-809 and VX-661
368 were similarly additive to 27ºC incubation of F508del-expressing cells, consistent with
369 previous reports demonstrating that VX-809 was unable to restore the thermostability of
370 F508del-CFTR [34]. The three correctors, which were additive to G550E and 4RK, had
371 an even greater effect when combined to low temperature. Although none of the
18
372 correctors was additive to R1070W at 37°C, additivity was observed for all three in
373 combination to low temperature for this revertant, which was however, less pronounced
374 for RDR01752. Altogether, these data indicate that despite the double correction effect
375 of revertants and compounds on F508del-CFTR, there is still scope for further
376 enhancement as indicated by low temperature data.
377 As with VX-809 and VX-661, RDR01752 was unable to overcome a Sec24-COPII-
378 ER export defect of the diacidic variant DD/AA (on WT-CFTR backbone). Low
379 temperature nevertheless enables DD/AA-CFTR to exit the ER through the conventional
380 ER-to-Golgi pathway, since the rescued DD/AA-CFTR was found to be fully-
381 glycosylated [17]. These data are compatible with the proposed mechanism for F508del-
382 CFTR by low temperature [17].
383 Although RDR01752 did not further enhance F508del-CFTR PM expression rescue
384 by VX-809 or VX-661 in the immunofluorescence assay, it demonstrated a trend to
385 increase the rescue of F508del-CFTR function when combined with VX-809 versus each
386 compound alone, albeit not significantly. This may be attributable to a weak potentiator
387 activity of RDR01752 on F508del-CFTR channels [14]. Notably, chronic VX-770
388 exposure reduced RDR01752-rescued CFTR PM expression in F508del-expressing cells,
389 as described for VX-809 and VX-661 [27,28]. However, chronic exposure of F508del-
390 expressing cells to a low free concentration of VX-770 prevents the negative effect on
391 VX-809-rescued CFTR [35]. Surprisingly, rescuing of CFTR PM expression by C18,
392 compound with a similar chemical structure to VX-809/VX-661 [36,37], was less
393 affected by chronic VX-770 exposure. In fact, C18 and VX-809 possibly have a different
394 MoA since in primary HBE cells, the former failed to rescue the trafficking mutant
395 A561E-CFTR, in contrast to VX-809 [26].
396 Because there are several CFTR mutations with the same trafficking defect as
397 F508del-CFTR, i.e., class II [2,3,30], but do not equally respond to the same CFTR
398 corrector [11,12,37-39], we investigated whether RDR01752 could rescue some of these
399 mutants. Functional assessment was initially performed by the FMP assay and then
400 confirmed in Ussing chamber measurements of CFTR activity in polarized FRT cells
401 stably expressing some of those CFTR mutations. As RDR01752 was demonstrated to be
402 a weak potentiator of F508del-CFTR channels [14], we tested its effect on R334W-CFTR,
403 which has minimal trafficking impairment but reduced channel conductance that still
404 allows for residual function [40]. A slight but non-statistically significant increase in
405 R334W-CFTR function was induced by RDR01752 whereas a greater effect was
406 observed with VX-809. Co-administration of RDR01752 and VX-809 did not further
407 increase R334W-CFTR function as compared to VX-809 alone, albeit a trend was
408 observed. We also tested RDR01752 on G85E and N1303K class II CFTR mutants which
409 are temperature-insensitive despite having trafficking defects like F508del [12,39].
410 N1303K was not rescued efficiently by RDR01752 or VX-809 when tested individually
411 or in combination, consistent with previous findings in HBE cells [26] and intestinal
412 organoids [11]. Regarding G85E, although it appeared to respond functionally to
413 RDR01752 in the FMP assay, this was not confirmed in the Ussing chamber
414 measurements. The lack of response of this mutant to several correctors has been
415 previously reported [12,38]. Altogether, these data demonstrate that both G85E and
416 N1303K trafficking defects are difficult to rescue and alternative correctors remain an
417 unmet need for these mutants.
418 In conclusion, these data show that RDR01752, like VX-809/VX-661, rescues
419 F508del-CFTR, albeit at lower efficiency, and like those two approved corrector drugs it
420 does not rescue the G85E and N1303K traffic mutants. Our studies with revertants, aimed
20
421 at understanding the MoA of this novel corrector, help explain such similarity in
422 pharmaco-therapeutic behavior. Indeed, the data suggest RDR01752 may share a binding
423 site on F508del-CFTR with VX-809 and VX-661, i.e., at the NBD1:ICL4 interface.
424 However, because RDR01752 is chemically distinct from VX-809/VX-661, it may have
425 an additional allosteric effect that causes its putative (weak) potentiating activity (see
426 Graphical abstract). The fact that RDR01752, like VX-809/VX-661, is additive to
427 correction by the revertants G550E and 4RK and also by low temperature indicate that
428 there is still scope for correctors to further increase the rescue of F508del-CFTR.
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27
553 7. Figure legends:
554 Figure 1 – RDR01752 rescues F508del-CFTR processing and PM expression. (A)
555 CFBE cells stably expressing F508del-CFTR were incubated for 24 h with DMSO
556 (negative control), VX-809 (3.7 µM), VX-661 (5 µM) or an increasing concentration of
557 RDR01752. (B) CFTR processing (C/B+C) was quantified and normalized to tubulin
558 levels (loading control). Data are shown as means + SD of 4 independent experiments.
559 Vs. DMSO: *P<0.05, **P<0.01. Vs. VX-809: #P<0.05, ##P<0.01. Statistical analysis was
560 performed using One-way ANOVA followed by Tukey’s post hoc test. (C) CFBE stably
561 expressing mCherry-Flag-F508del-CFTR were incubated for 48 h with DMSO (negative
562 control), VX-809 (3.7 µM) or an increasing concentration of RDR01752. (D)
563 Immunostaining was performed and fluorescence images of extracellularly exposed Flag-
564 tags were quantified to determine CFTR PM expression. The deviation score relative to
565 negative control (DMSO) was calculated and plotted. Data are shown as means + SD of
566 4 independent experiments. Vs. DMSO: *P<0.05. Vs. VX-809: #P<0.05. Statistical
567 analysis was performed using two-tailed unpaired Student’s t-test.
568 Figure 2 – RDR01752 rescues F508del-CFTR function. (A) Representative cell
569 fluorescence recording acquired with a microplate reader. CFBE cells stably co-
570 expressing F508del-CFTR and the HS-YFP were incubated for 24h with DMSO
571 (vehicle), VX-809 (3.7 µM) or increasing concentrations of RDR01752. Cells were then
572 acutely (30 min) stimulated with Fsk (20 µM) and Gen (50 µM). (B) CFTR activity was
573 quantified based on the rate of YFP quenching and normalized to the negative control
574 (DMSO, dashed line). Data are shown as means + SD of 4 independent experiments. Vs.
575 DMSO: *P<0.05, ** P<0.01. Vs. VX-809: #P<0.05. Statistical analysis was performed
576 using One-way ANOVA followed by Tukey’s post hoc test. (C-F) Monolayers of CFBE
577 cells stably expressing F508del-CFTR were incubated for 24 h with (C) DMSO (negative
28
578 control), (D) VX-809 (3.7 µM), or (E) RDR01752 (10 µM). Original Ussing chamber
579 (open-circuit) recordings depicting transepithelial voltage measurements (Vte). There is
580 an absence of response in cells treated with DMSO, while negative deflections are
581 observed in cells treated with VX-809 or RDR01752 following the application of
582 Fsk+IBMX and genistein, which are reverted by application of Inh172. (F) Data are
583 expressed as Isc calculated from voltage deflections obtained for the responses to
584 Fsk+IBMX+Gen. Data are shown as means + SD of 3 independent experiments. Vs.
585 DMSO: *P<0.05, **P<0.01. Vs. VX-809: #P<0.05. Statistical analysis was performed
586 using One-way ANOVA followed by Tukey’s post hoc test.
587 Figure 3 – Intestinal organoids (F508del/F508del) respond positively to RDR01752.
588 (A) Bright-field images of organoids incubated for 24 h with DMSO (negative control),
589 VX-809 (3.7 µM), VX-661 (5 µM) or RDR01752 (10 µM) and acutely stimulated with
590 forskolin (Fsk, 0.128 µM) with VX-770 (3 µM) or genistein (Gen, 50 µM). (B) Data of
591 FIS of organoids are expressed as the absolute area under the curve (AUC; baseline =
592 100%, t = 60 min, 0.128 µM Fsk). Data are shown as means + SD of 3 independent
593 experiments. Absence of bars indicates there was no swelling (NS). Vs. DMSO: *P<0.05,
594 **P<0.01, ***P<0.001. Statistical analysis was performed using two-tailed unpaired
595 Student’s t-test.
596 Figure 4 – RDR01752 increases the rescue of F508del-CFTR PM expression in low
597 temperature and in cells expressing in cis the genetic revertants G550E and 4RK,
598 but not in R1070W or the null-traffic DD/AA variant. CFBE stably expressing
599 mCherry-Flag-CFTR (WT, F508del, DD/AA variants or carrying G550E, R1070W, 4RK
600 in cis with F508del) were incubated for 24 h with DMSO (negative control), RDR01752
601 (10 µM), VX-809 (3.7 µM) or VX-661 (5 µM) and then maintained for additional 24h at
602 (A) 37°C or (B) in low temperature (27°C). The deviation scores relative to negative
29
603 control (DMSO at 37°C) were calculated from fluorescence images of extracellularly
604 exposed Flag-tags and plotted to determine CFTR PM expression. Data are shown as
605 heatmaps of 6 independent experiments. Statistical analysis was performed using two-
606 tailed unpaired Student’s t-test.
607 Figure 5 – RDR01752 is not additive to VX-809 or VX-661 in rescuing F508del-
608 CFTR traffic. (A) CFBE stably expressing mCherry-Flag-F508del-CFTR were
609 incubated for 48 h with the following compounds individually or in combination: DMSO
610 (negative control), RDR01752 (10 µM), VX-809 (3.7 µM), VX-661 (5 µM), C18 (5 µM)
611 and VX-770 (3 µM). (B) Immunostaining was performed and deviation scores relative to
612 negative control (DMSO) were calculated from fluorescence images of extracellularly
613 exposed Flag-tags and plotted to determine CFTR PM expression. Data are shown as
614 means + SD of 4 independent experiments. Vs. single corrector (white bars): *P<0.05,
615 ***P < 0.001. n.s.: no significant. Statistical analysis was performed using two-tailed
616 unpaired Student’s t-test.
617 Figure 6 – Assessment of RDR01752 effects on rare CFTR mutants by FLIPR
618 membrane potential (FMP) assay. FRT cells stably expressing CFTR variants (WT,
619 G85E, R334W, T338I, R347P, F508del, V520F, S549F, G551D, M1101K or N1303K)
620 were incubated for 24 h with DMSO (negative control), RDR01752 (10 µM) or VX-809
621 (1 µM). FMP assay was performed to monitor membrane depolarization induced by
622 stimulation Data are shown as means + SD of 3 independent experiments. Vs. DMSO
623 (black bar for each CFTR variant): *P<0.05. Statistical analysis was performed using
624 two-tailed unpaired Student’s t-test.
625 Figure 7 – Effect of RDR01752 and VX-809 individually or in combination on
626 functional rescue of CFTR carrying F508del, G85E, N1303K or R334W. (A)
627 Monolayers of FRT cells stably expressing CFTR variants (F508del, G85E, N1303K or
30
628 R334W) were incubated for 24 h with DMSO (negative control), VX-809 (1 µM) and
629 RDR01752 (10 µM) alone or combined. Representative recordings of Isc measurements
630 of Ussing chamber for each CFTR mutant. CFTR currents were stimulated using
631 forskolin (FSK; 10 µM) and genistein (GST; 50 µM) and inhibited by CFTRinh -172 (172;
632 10 µM). ATP (100µM) was added at the end of each experiment as a positive control for
633 viability. (B) Data are represented as mean increase in Isc induced by FSK+GST. Data
634 are shown as means + SD of 3-6 independent experiments. Vs. DMSO: *P < 0.05, **P <
635 0.01. Statistical analysis was performed using two-tailed unpaired Student’s t-test.
636 Credit Author Statement
637 Conceptualization: MLP, JWH, MDA. Data acquisition: MLP, IALS, MJT, GWC, ES,
638 DYT, NP. Data analysis and interpretation: MLP, IALS, MJT, GWC, ES, DYT, NP,
639 JWH, MDA. Funding acquisition: DYT, JWH, MDA. Writing of first draft: MLP, MDA.
640 All authors read and approved the final version of the manuscript.