Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity

Article abstract of DOI:10.1016/j.ejmech.2020.112116

Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC₅₀ close to 1 μM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.

Full text of DOI:10.1016/j.ejmech.2020.112116

Journal Pre-proof
Targeting different binding sites in the CFTR structures allows to synergistically
potentiate channel activity
Lionel Froux, Ahmad Elbahnsi, Benjamin Boucherle, Arnaud Billet, Nesrine Baatallah,
Brice Hoffmann, Julien Alliot, Renaud Zelli, Wael Zeinyeh, Romain Haudecoeur,
Benoit Chevalier, Antoine Fortuné, Sandra Mirval, Christophe Simard, Pierre Lehn,
Jean-Paul Mornon, Alexandre Hinzpeter, Frédéric Becq, Isabelle Callebaut, Jean-Luc
Décout
PII:
S0223-5234(20)30083-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112116
EJMECH 112116
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 August 2019
Revised Date: 24 January 2020
Accepted Date: 3 February 2020
Please cite this article as: L. Froux, A. Elbahnsi, B. Boucherle, A. Billet, N. Baatallah, B. Hoffmann, J.
Alliot, R. Zelli, W. Zeinyeh, R. Haudecoeur, B. Chevalier, A. Fortuné, S. Mirval, C. Simard, P. Lehn, J.-
P. Mornon, A. Hinzpeter, Fréé. Becq, I. Callebaut, J.-L. Décout, Targeting different binding sites in the
CFTR structures allows to synergistically potentiate channel activity, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112116.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Masson SAS.

Targeting different binding sites in the CFTR structures allows to
synergistically potentiate channel activity
a,1
b,1
c,1
a
Lionel Froux , Ahmad Elbahnsi , Benjamin Boucherle , Arnaud Billet , Nesrine
d,e
b,3
c,4
c,5
c,6
Baatallah , Brice Hoffmann , Julien Alliot , Renaud Zelli , Wael Zeinyeh , Romain
c
d,e
c
a
a,7
Haudecoeur , Benoit Chevalier , Antoine Fortuné , Sandra Mirval , Christophe Simard ,
f
b
d,e,#
a,2
Pierre Lehn , Jean-Paul Mornon , Alexandre Hinzpeter , Frédéric Becq , Isabelle
b,2*
c,2
Callebaut , Jean-Luc Décout
a
Laboratoire Signalisation et Transports Ioniques Membranaires, Université de
Poitiers, 86073, France.
Sorbonne Université, Muséum National d’Histoire Naturelle, UMR CNRS 7590,
b
Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005 Paris,
France.
c
d
e
f
Univ. Grenoble Alpes, CNRS, DPM, 38000 Grenoble, France
INSERM, U1151, Institut Necker Enfants Malades, INEM, Paris, France
Université de Paris, Paris, France
INSERM UMR1078, IBSAM, Université de Bretagne Occidentale, Brest, France
1
2
*
These authors contribute equally
These authors contribute equally
Corresponding author: IMPMC, UMR7590 CNRS-Sorbonne Université, Case 115, 4 place
Jussieu, 75252 Paris Cedex 05, France ; e-mail : isabelle.callebaut@sorbonne-universite.fr
3
4
5
6
7
Present address : Iktos, Paris, France
Present address : Technologie Servier, Orléans, France
Present address : Institut de Recherches cliniques de Montréal, Montréal, Canada
Present address : ISPB-Faculté de Pharmacie, Université de Lyon 1, Lyon, France
Present address : Université de Caen, Caen, France
Summary declaration of interest: The authors declare the following competing financial
interest related to their patent: Compounds for treating cystic fibrosis. I. Callebaut, J.-P.
Mornon J.-L. Décout, F. Becq, Pierre Lehn, Brice Hoffmann, B. Boucherle, A. Fortuné, R.
Haudecoeur, C. Boinot, WO 2016 087665 A2 20160609.
1

A
BSTRACT
Recent evidence shows that combination of correctors and potentiators, such as the drug
ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic
Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated
in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of
identifying a broad panel of specific binding mode modulators, occupying several distinct
binding sites at structural level. Here, we identified two small molecules, SBC040 and
SBC219, which are two efficient cAMP-independent potentiators, acting at low
concentration of forskolin with EC close to 1 µM and in a synergic way with the drug VX-
5
0
7
70 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested
potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently
proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.
Keywords: CFTR, modulator, combinatorial therapies, 3D structure, Ussing chamber,
halide-sensitive fluorescence
2

1. INTRODUCTION
Mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR, ABCC7) gene
cause Cystic Fibrosis (CF), an autosomal recessive disease affecting approximately 80,000
people worldwide and characterized by progressive lung disease [1]. The CFTR protein is an
epithelial anion channel, made of: (i) four well-folded domains (the Membrane-Spanning
Domains MSD1 and MSD2, as well as the Nucleotide-Binding Domains NBD1 and NBD2), (ii) a
regulatory (R) region showing intrinsic disorder and whose phosphorylation regulates
channel activity, and finally (iii) N- and C-terminal segments, which mediate interaction with
cellular partners [2]. More than 2,000 mutations have been reported so far, conferring a
variety of molecular defects. The current classification of mutations into six different classes
can be extended, by taking into account the observation of combinatorial defects for some
of them and their susceptibility to modulators ([3]. Accordingly, the most common F508del
mutation (a deletion of Phe508 in NBD1), impairs CFTR domain folding and folding
cooperativity, leading to premature degradation (hallmark of class II mutations), but also
leads to gating defect (as for class III mutations), as well as to instability at the plasma
membrane (class VI mutations) [3]. Remarkably, it is now generally agreed that these defects
can be rescued by combination of pharmacotherapies addressing single defects (i.e.
correctors and potentiators acting respectively on protein maturation and channel function).
Applying a corrector such as VX-809 (Lumacaftor) alone has little efficiency, while combining
it to the potentiator VX-770 (Ivacaftor) improves function [4]. However, there is only modest
clinical benefit [5] possibly due to remaining uncorrected single defects and increased
turnover rate upon chronic VX-770 treatment [6, 7]. This therapeutic ceiling of first-
generation correctors has led to the development of novel screening strategies for
identifying second-generation correctors that could be used synergistically, in combination
with the first ones [8]. Hence, it has recently been shown that combination of correctors
exhibiting individually low rescue of F508del CFTR but acting through different mechanisms
can robustly restore the functional expression of CFTR in human airway cells [9, 10].
Accordingly, one can distinguish: (i) type I correctors, targeting NBD1/MSD1 and
NBD1/MSD2 interfaces, (ii) type II correctors, targeting NBD2 and/or its interfaces and (iii)
type III correctors, acting on NBD1 folding/stability. This synergic action of correctors
strongly suggests that several distinct binding sites are occupied within the CFTR protein.
3

Occupation of these sites may moreover lead to a general rescue of mutated proteins
through allostery [10]. Along these lines, a triple combination of pharmacological
chaperones (VX-809, RDR1 and MCG1516A) has also recently been reported, the three
correctors having however been proposed to interact with NBD1 [11]. The importance of
binding multiple sites for correction is now also underscored by the recent report of
improved clinical efficiency when adding to ivacaftor (VX-770) + tezacaftor (VX-661)
bitherapy new generation correctors (VX-445 and VX-659) targeting distinct sites of the
protein [12, 13]. The search for alternative potentiators, which is following the same
rationale of combinatorial therapy, remains required for mutations that appear refractory to
treatment by single potentiators alone or for the use in combination with correctors, with no
interference with the action of the latter or effect on protein stability [14-17].
The development of these combinatorial approaches highlights the importance of having a
comprehensive inventory of the modulator binding sites within the CFTR protein 3D
structure and identifying a broad panel of modulators. Potential binding sites, which may be
targeted by drugs, have already been proposed (e.g. [18-24]), by considering models of 3D
structures of the MSD:NBD assembly [25-27] and high-resolution cryo-EM 3D structures of
full length CFTR in different closed conformations [24, 28-30]. Recently, a cryo-EM study has
given a first view of a likely binding site for ivacaftor and GLPG1837, another CFTR
potentiator, within the MSDs. However, the channel in the complex is still in a near open,
closed conformation, although phosphorylated and ATP-bound [31].
In the present work, we have developed a series of small molecules (called SBC for Small
Binders of CFTR), initially designed for interacting with an obvious target site on CFTR, i.e.
filling the pocket known to be present in the altered ICL4:NBD1 inter-domain interface in the
F508del 3D structure. These molecules did however not display any obvious corrector
activity, as expected from their design, but a potentiator activity was found in both F508del
and wild-type CFTR, and variable effects were observed on several class III mutations
sensitive to VX-770. A synergic effect was observed with VX-770 in comparison to SBC alone
and VX-770 alone, suggesting that SBCs and VX-770 act through different sites and
mechanisms. These features, associated with the SBC ATP-like purine scaffold, led to the
hypothesis, supported by molecular dynamics (MD) simulations, that the SBC molecules act
4

on the CFTR protein by occupying its ATP-binding site(s). This highlights the interest of such
molecules as tools for a pharmacotopological analysis of CFTR, allowing to better understand
its mechanisms of action and opening new way for rational improvement of CFTR
pharmacology.
2. RESULTS
2
.1. Synthesis of a series of small CFTR binder compounds (SBCs)
We hypothesized that small molecules designed to occupy potential drug binding sites
would lead to the discovery of new modulators of normal and defective CFTR chloride
channel functions. A first series of small molecules was designed, by taking advantage of a
model of the CFTR MSDs:NBDs assembly available at the beginning of this work [26], in
which the molecules occupy the room left by the F508 deletion at the interface between
NBD1 and ICL4 (F508del pocket, Figure S1 (supporting information)). We chose to graft a
phenyl group mimicking that of the F508 residue at the N9 of a purine bicycle scaffold, which
is incorporated in many drugs and drugs candidates, for example, the cyclin-dependent
protein kinase inhibitor (R)-Roscovitine (Seliciclib) [32]. The purine bicycle is made of five
carbon and four nitrogen atoms well distributed in the bicyclic system. This distribution
makes it a central scaffold in the search for pharmacological and therapeutic agents, being
well adapted for H-bond formation, and the introduction of chemical functions and
recognition elements at different positions on carbon and/or nitrogen atoms [33, 34]. In
order to increase the solubility in water and to allow H-bonding from the phenyl ring, a
carboxamido group CONH was placed on the phenyl ring and, thus, the SBC001 structure
2
appeared to be an interesting starting molecule allowing further chemical modifications by
substitution of the C2 and C6 chlorine atoms. This structure was modified according to
molecular docking in order to establish stronger interactions with the target sites and to
preserve flexibility. Ten purines SBC001, 005, 040, 068, 069, 071, 183, 200, 219 and 231
carrying at position 9 a 4’-carboxamidophenyl group (Fig. 1 and scheme 1 (A to I)) were
hence designed and, then, were chemically synthesized (Details of the synthesis are given in
9
Figure S2 (supporting information)). SBC001 was prepared by N -arylation of 2,6-
dichloropurine in 16% yield through the Chan-Evans-Lam coupling reaction with (4-
carbamoylphenyl)boronic acid and copper(II) [35]. Subsequent chloride substitution allowed
5

the introduction of selected specific groups at position 6 and led to the 2-chloropurines
SBC005 and SBC068. Preliminary chloride substitution from 2,6-dichloropurine afforded the
9
2
-chloropurines SBC040 and 219 through N -arylation with (4-carbamoylphenyl)boronic
9
acid/copper(II) and with 4-iodobenzamide/copper(I) [36], respectively. These methods of N -
arylation were used to prepare the 6-aminopurines SBC071 and 069, respectively, directly
from 2,6-diaminopurine and N,N-(bis(tert-butoxycarbonyl)adenine. The synthesis of the
purines SBC183, 200 and 231 was achieved from 5-amino-4,6-dichloropyrimidine and 2-
amino-4,6-dichloro-5-formamidopyrimidine [37]. More recently, we developed a one-pot
synthesis of highly functionalized purines from 4,6-dichloropyrimidines allowing for example
the rapid and efficient preparation of SBC001 in 86% yield [38-40].
2
.2. Characterization of the SBC activity
2
.2.1. SBCs are not correctors of F508del-CFTR maturation
We first evaluated the effect of SBCs on F508del-CFTR maturation in Western-Blot
experiments using stably transfected Hela cells expressing F508del-CFTR and an antibody
directed against CFTR protein. The biochemical profiles of F508del-CFTR were similar in the
absence or presence of any of the SBCs, tested as shown in Figure S3 (supporting
information). We used VX-809 as positive control to confirm the appearance of the mature
form of CFTR (C band), an indicator of F508del-CFTR maturation correction. Thus, contrary to
VX-809, SBCs are not correctors of F508del-CFTR maturation.
2
.2.2. SBCs potentiate F508del- and wild-type (WT)-CFTR currents:
The ten purines synthesized were then screened for a possible potentiator activity using
Ussing chamber experiments on CFBE F508del-CFTR cells corrected 24 h at 27°C to partially
restore the membrane localization of the mutant. Compounds were sequentially tested at
three concentrations: 10, 50 and 100 µM with a minimal pre-activation of CFTR with
forskolin, an adenylate cyclase activator producing intracellular cAMP (0.1 µM; hereafter
noted fsk) (Fig. 2A and Fig. 2B). We identified three compounds - namely, SBC001, SBC040
and SBC219 - able to elicit a significant CFTR 172-sensitive increase of the transepithelial
inh
short-circuit current (hereafter noted Isc) over fsk effect, others being inactive or producing
very weak effects. Since SBC040 and SBC219 had the strongest effects, we considered them
as leaders and determined their half-maximal effective concentration (EC ) on CFBE
5
0
6

F508del-CFTR cells (Fig. 2C). With fsk (0.1 µM) in the recording chamber, the EC values
5
0
measured were 1.51 ± 0.21 µM (n= 4) for SBC040 and 0.87 ± 0.11 µM (n= 6) for SBC219.
Consistent with data published by other groups [41], EC value measured for VX-770 was
5
0
almost 100 fold lower on these cells under our conditions (11.7 ± 1.5 nM; n= 6).
As the effects of SBC040 and SBC219 could be due to an augmentation of intracellular cAMP
levels, we measured the intracellular cAMP concentration in CFBE F508del-CFTR cells after a
1
5-minute incubation with each SBC at concentrations leading to the maximal potentiator
effect on F508del-CFTR (30 µM for SBC040 and 10 µM for SBC219). We did not detect any
significant variation of intracellular cAMP levels after acute addition of the two leader
compounds compared to solvent (Fig. 2D), suggesting their potentiator effect is not
mediated by cAMP-PKA pathway activation.
Results obtained on CFBE F508del-CFTR were confirmed using automatic whole-cell planar
patch-clamp on Hela cells stably expressing F508del-CFTR and corrected 24 h at 27°C (Fig. 3).
We recorded a time and voltage independent current fully inhibited by 10 µM of CFTR 172
inh
after the simultaneous addition of fsk (1 µM) + SBC040 (30 µM) or fsk + SBC219 (10 µM). The
current densities recorded in the presence of SBC040 and SBC219 (at +40 mV: 38.2 ± 6.6
pA/pF (n=21) for SBC040 and 53.3 ± 9.6 pA/pF (n= 10) for SBC219) were significantly larger
than the current density elicited by fsk alone (15.6 ± 1.9 pA/pF (n= 15) at +40mV), confirming
the ability of SBCs to potentiate F508del-CFTR currents (p= 0.0011 for SBC040 and p= 0.0008
for SBC219; two-tailed Mann-Whitney test). Also, by comparison, the efficiency of SBC040
was similar to the clinically approved potentiator VX-770 effect (current density at +40 mV:
3
7.5 ± 5.7 pA/pF (n= 23) while SBC219 presented a stronger potentiation.
We also detected a potentiator effect on wild-type (WT)-CFTR in Hela cells with both SBCs,
with a stronger effect of SBC219 (Figure S4 (supporting information)). We did not observe
major cytotoxic effects of SBCs as shown Figure S5 (supporting information) in HeLa cells
incubated for 24h with SBC040 or SBC219 at 1 and 10 µM. Of note is that the SBCs display
activity at submaximal concentration of forskolin (0.1 µM) on F508del-CFTR, the effect
disappearing at higher concentration (Figure S6 (supporting information)). This behavior
contrasts with the VX-770-mediated potentiation of CFTR, which is dependent on
7

phosphorylation level [42] (see below and discussion). Several CFTR potentiators are known
to interfere with the correction of F508del-CFTR maturation defect by VX-809 or VX-661 [6,
7
]. Preliminary data obtained by western-blot suggest that SBC040 have a negative effect on
VX-809 correction, in the same range as VX-770 (data not shown).
2
.2.3. Effect of SBC040 and SBC219 on G551D and G1349D mutations, and class III
mutations
Then, we tested the potentiator effect of the two SBC leaders on two major class III CFTR
mutants: G551D and G1349D, which are sensitive to VX-770 [43], the potentiator used to
treat the resulting cystic fibrosis. These two amino acids occupy similar positions in the ABC
signature of NBD1 and NBD2, respectively, bordering the canonical and non-canonical ATP-
binding sites, respectively. The SBC effect was first evaluated using conventional whole cell
patch-clamp on Hela cells transiently transfected with a pEGFP-CFTR-G551D or -G1349D (Fig.
-
4
). While no potentiation of CFTR Cl currents was observed with SBC040 on both mutants
(Fig. 4 top panels), application of 10 µM SBC219 elicited a 2.6 fold potentiation of fsk pre-
-
activated CFTR current (Fig. 4 bottom panels) for both mutants. These Cl currents recorded
were fully inhibited by the CFTR 172. However, the potentiation of CFTR current by SBC219
inh
was not maximal on these mutants since the current elicited when using SBC219 remained
lower than when the investigational CFTR potentiator genistein was used.
We evaluated the effect of SBCs on class III CFTR mutants expressed in HEK293 cells using a
halide sensitive YFP assay. In control WT-CFTR expressing cells, SBC040 and SBC219
potentiated CFTR activity at both 10 µM and 50 µM to a slightly lower level to that observed
with VX-770 (Fig. 5A). G551D and G1349D were found to be responsive to VX-770
treatment, with higher correction achieved for G1349D as compared to G551D. A stronger
effect was observed for SBC219 on G1349D compared to SBC040 (Fig. 5A). Interestingly, the
effect of both SBC040 and SBC219 were more pronounced on G551S CFTR (Fig. 5A),
suggesting an effect of the length or polarity of the side chain introduced in the canonical
binding site. The sensitivity to SBC040 and SBC219 of two additional class III mutations,
sensitive to VX-770 and located within NBD2, was also tested. The first one (G1244E) is
located within the canonical ATP-binding site, in front of NBD1 G551D. This mutation
showed similar low responses regarding the effect of SBC040 and SBC219 (Fig. 5A). In
contrast, S1255P, which is located farther from the canonical binding site, displays responses
8

to both SBC040 and SBC219 (Fig. 5A). Finally, the sensitivity to SBC040 and SBC219 of a VX-
70-sensitive class III mutation located in ICL3 (G970R) was also tested. This mutation
7
showed increased levels of responses with SBC040 and SBC219, with a level of potentiation
reached by SBC219 at 50 µM similar to that of VX-770 at 10 µM (Fig. 5A).
2
.2.4. Potentiator effect of SBCs is synergic with VX-770 effect and compatible with VX-809
correction
We then addressed the question of a potential interaction with correctors or other
potentiators. We therefore compared the effects of SBC040 and SBC219 to the effect of VX-
7
70 on CFBE F508del-CFTR cells treated 24 h with VX-809 before Ussing chambers
experiments. We also added VX-809 (10 µM) to the apical solution 5 min before protocol
starting to ensure the presence of the molecule during the whole experiment. During these
experiments, SBCs were added either before or after VX-770 and were used at
concentrations leading to their maximal effect. We also added fsk (0.1 µM) to pre-activate
CFTR before SBC or VX-770 addition (Fig. 6A). When focusing on the effect of each
compound added alone, the variation of Isc (noted ΔIsc and defined as the variation of Isc
induced by the addition of a compound to the apical membrane) was significantly lower
-
2
-2
after SBC addition (1.2 ± 0.2 µA.cm for 040 and 0.91 ± 0.11 µA.cm for 219, n= 7) than after
-
2
VX-770 addition (3.0 ± 0.2 µA.cm , n= 14). However, when adding SBC and VX-770
-
2
sequentially, ΔIsc increased when compared to VX-770 alone (5.1 ± 0.3 µA.cm with 040 or
-
2
-2
6
.3 ± 0.5 µA.cm with 219 vs. 3.0 ± 0.2 µA.cm alone, n=14) (Fig. 6B) suggesting an additivity
or a synergy between SBC and VX-770 effects. Of note is that the combination of SBCs and
VX-770 at low forskolin concentration leads to effects greater than those obtained for VX-
7
70 alone at high forskolin concentration (Figure S6 (supporting information)). This effect,
even at lower phosphorylation level of CFTR, demonstrates the interest of SBCs despite the
absence of effect alone at high forskolin concentration.
We then focused the data analysis on the repartition of the different effects, depending on
the order in which the compounds were added (Fig. 6C and Fig. 6F). As expected, we noticed
a significant effect of the compounds (p< 0.0001 for both SBCs; n= 7) but not of the order in
which the compounds were added on ΔIsc (p= 0.45 for SBC040 and p= 0.88 for SBC219; n=7).
The statistical analysis also returned a positive result for interaction significance, suggesting
9

the order in which the compounds were added could have influenced their individual
effects. This hypothesis is supported by the fact that there is a significant difference between
VX-770 and SBC effects when SBCs were added before VX-770 but not when SBCs were
added after VX-770 (for both SBCs: p< 0.001; n= 7). This suggests a possible synergy between
SBCs and VX-770 effects besides additivity. To confirm this, we used the same data set and
focused our attention on the amplitude of the VX-770 effect, whether VX-770 was added
alone or after SBC040 or SBC219. The same comparison was done between the effect of
SBC040 or SBC219 alone or after the addition of VX-770. We noticed a significant
-
2
augmentation of VX-770-induced ΔIsc when SBC040 (Fig. 6D; 2.82 ± 0.22 µA.cm vs 3.84 ±
-
2
-2
-
0
.26 µA.cm ; n= 6-7; p= 0.035) or SBC219 (Fig. 6G; 3.12 ± 0.34 µA.cm vs 5.45 ± 0.59 µA.cm
2
;
n=7; p=0.002) was added before VX-770 and of SBC-induced ΔIsc when VX-770 was added
-
2
-2
before SBC040 (Fig. 6E; 1.21 ± 0.20 µA.cm vs 2.57 ± 0.44 µA.cm ; n= 7; p= 0.007) or SBC219
-
2
-2
(
Fig. 6H; 0.91 ± 0.11 µA.cm vs 3.02 ± 0.48 µA.cm ; n= 7; p= 0.0006), even if this effect was
larger for SBC- than for VX-770-induced ΔIsc. Taken together, these results show that SBCs
are able to potentiate F508del-CFTR current in the presence of VX-809 and that the effects
of SBCs and VX-770 are synergic.
The synergy of SBCs with VX-770 was also evaluated using the YFP halide sensitive assay. No
further potentiation was observed for WT-CFTR, using either SBC combination or VX-770
with either SBC (Fig. 5B). This was in contrast to results obtained with G551D-, G970R- and
G1349D-CFTR (Fig. 5B). Both SBCs indeed showed stronger effect with VX-770 on these
three mutated proteins, enabling to restore WT levels of CFTR function. Moreover, these
results suggest a synergy between SBC and VX-770 effects on G551D-, G970R- and G1349D-
CFTR since the effect of SBC040 or SBC219 combination with VX-770 was greater than the
sum of SBCs and VX-770 individual effects on these mutants.
2
.3. Mapping SBC potential binding site(s)
As the two SBC040 and SBC219 act in a synergistic way with VX-770, we hypothesized that
they may bind site(s) distinct from this/those occupied by VX-770, for which several
evidences now indicate potential binding sites within the MSDs (see Discussion). As the SBCs
act on WT-CFTR, we also ruled out a potential binding site at the interface between ICL4 and
NBD1 (F508del pocket), as initially targeted on the model of F508del-CFTR 3D structure.
1
0

Given the SBC general scaffold and the fact that SBC040 has no or poor potentiation effect
on the G551D and G1349D-CFTR contrary to WT-CFTR (see above), a consistent hypothesis
for the SBC-binding sites (at least SBC040) should be within one or both ATP-binding sites. To
test this hypothesis, we first docked SBC040 and SBC219 in the two free ATP-binding sites
within the model of the 3D structure of the wild-type CFTR MSDs:NBDs assembly in an open
conformation, as previously published [25]. We used this model rather than the more recent
cryo-EM 3D structures of phosphorylated, ATP-bound zebrafish and human CFTR [29, 30], as
these last ones are yet in a closed conformation, even when solved in complex with
ivacaftor, as recently reported [31]. The reliability of the whole models has however been
supported by comparison with these experimental 3D structures [44]. The conformations of
the NBD1:NDB2 assembly and of their interfaces with the ICLs are moreover highly similar,
as illustrated in Figure S7 (supporting information).
The results of this docking on the CFTR 3D structure model were supported by an automatic
docking performed using the Autodock Vina and AutoDockTools (45 % of the 20 best hits in a
docking experiment performed on NBDs felt within the ATP-binding sites). Molecular
Dynamics (MD) simulations on SBC040 and SBC219 docked within the canonical and non-
canonical ATP-binding sites were then performed over periods > 100 ns for testing their
adaptation to/stability in these sites and identifying of critical residues. Overall stability of
the trajectories is observed from 50-60 ns, as appreciated by stable RMSD values, not only
for the wild-type constructs, but also for F508del (simulation performed at 300 K) (Figure S8
(supporting information)). Notably, in the bound complexes, the CFTR MSDs:NBDs assembly
displays features of the open conformation, as illustrated for instance with the presence of
salt bridges between E267 (ICL2) and R1060 (ICL4) [45, 46] and between R352 (TM6) and
D993 (TM9) [47, 48] (data not shown). During simulations, the SBC molecules stayed at the
level of ATP-binding sites, but their positions differently evolved according to the considered
ligand and ATP-binding site, with significant plasticity observed to accommodate the ligand
to the binding site. Maps of contacts between SBC and CFTR amino acids were established
along simulations, allowing to accurately define amino acids participating in binding and
identify the most representative conformations by cluster analysis (See Material and
Methods, Figure S9 (supporting information)). SBC features in the canonical and non-
canonical binding sites of WT-CFTR (most populated clusters) are detailed in Fig. 7 and
1
1

Figure S10 (supporting information). In the canonical binding site, SBC040 occupies the ATP
binding site, with contacts limited to NBD1 and NBD2 residues. In NBD1 are involved amino
acids of the ABC signature (of which Q552 which makes a hydrogen bond with the SBC040 O
amide) and other amino acids of the α-subdomain (of which F533, L541 and T547). In NBD2,
are principally involved amino acids of the Walker A motif, of which the G1247 N main atom
making an H-bond with the SBC040 N amide. In contrast, SBC219 occupied an upper
position, with several contacts with amino acids from ICL2 (of which Q270) and ICL3
(including M961 and N965). Of note is the NBD2 A loop Y1219 (which stacks the ATP adenine
ring) makes critical contacts with the purine ring of both SBC040 and SBC219 in the canonical
ATP-binding site. In addition, in SBC219, two N atoms of the purine ring make H bonds with
the Y1219 main chain oxygen atom. Notably, there was significant reorganization of the
residues within the ATP-binding site to accommodate ligands, as assessed by calculation of
backbone “cross” RMSD on the conserved motif of the ATP-binding sites (Figure S11
(supporting information)). Interestingly, the position of SBC040 and SBC219 at the level of
the ATP-binding site after MD simulation are similar in the wild-type protein and in the
F508del construct, involving similar contacts (Figure S12 (supporting information)). The
same situation is observed between wild-type CFTR and G551D CFTR (Figure S13 (supporting
information)). At the level of the non-canonical ATP-binding site (Fig. 7), which is covered by
the large regulatory insertion (RI), SBC040 and SBC219 principally make contacts with NBD1
(and very few with NBD2). As at the level of the canonical binding site, the A loop aromatic
side chain (W401) is also involved in SBC binding. However, the two SBC molecules are
positioned differently, SBC219 being located in an upper position, in the vicinity of the
NBDs:ICLs interface. Again, there was significant reorganization of the residues within the
ATP-binding site to accommodate ligands, as assessed by calculation of backbone cross
RMSD on the conserved motif of the ATP-binding sites (data not shown).
Based on the performed simulations, free energy binding values were estimated according
to Molecular Mechanics/Generalized Born Surface Area (MMGBSA) calculations. Values
obtained for SBC219 in both the canonical (-35.4 ± 4.5 kcal/mol) and non canonical (-35.3 ±
4
3
.1 kcal/mol) are similar and greater than those obtained for SBC040 (-15.3 ± 4.9 and -21.3 ±
.2 kcal/mol, respectively). Considering these values thus does not allow to favor one site
over another for SBC binding.
1
2

3. DISCUSSION
In light of the current efforts developed today towards combinatorial pharmacotherapy,
mapping in a comprehensive way the different possible binding sites of modulators which
may act in an additive or synergic way, is of outmost importance. However, except from a
possible binding site for ivacaftor recently highlighted within the MSDs using cryoelectron
microscopy [31], no direct experimental evidence describing CFTR modulator-binding sites is
available, which can be considered to explain the molecular mechanisms involved in the
modulation and to provide insights for improving existing molecules or designing new ones.
Several observations have however led to suggest different potential sites for correctors and
potentiators. The lack of two secondary structure elements (strand s5 and helix h2) in CFTR
NBD1 relative to standard ABC NBDs leads to an imperfect ball-and-socket joint at the
interface between NBD1 and MSDs (more exactly in the space separating intracellular loops
ICL1 (from MSD1) and ICL4 (from MSD2)), offering a potential binding site that could be
targeted by drugs [19, 24]. Another potential binding site is provided at the interface
between ICL4 and NBD1, when F508 is absent (F508del pocket). This site has logically been
explored as a potential corrector-binding site [20] and overlaps the putative binding site of
some correctors identified by virtual screening using the F508del NBD1 3D structure [22].
This F508del pocket was also targeted for the design of the SBCs discussed herein, but it has
however been discarded as their actual binding site, due to the potentiation activity of the
compounds also observed on the wild-type CFTR protein. In vitro studies have provided
information about the mechanism of action of VX-809, which stabilizes MSD1, involving
amino acids Phe374 and Leu375, in the early steps of co-translational folding [49, 50]. Based
on this information, a potential VX-809-binding site has been identified by molecular docking
at the bottom of MSD1, including Phe374 and Leu375 at the end of TM6 ([21] and our
unpublished results). Two studies have also proposed that Lumacaftor can bind NBD1 [11,
5
1]. Finally, putative binding sites of type III correctors (acting on NBD1 folding/stability) can
be searched at the surface of (or buried within) NBD1. These investigations are encouraged
by the effects of stabilizing mutations [52] or stabilizing nanobodies [53], but so far, NBD1
binders have however shown either (i) low affinity (e.g. CFFT-01 and BIA (bromoindole-3-
acetic acid), which both bind to an inner NBD1 pocket) [54]) or (ii) no activity on full length
1
3

F508del CFTR [16], precluding their development as correctors [55]. Higher affinity type III
correctors have very recently been proposed (4172 compound in [10]), but their NBD1
binding sites remain to be unveiled; it is also the case for the c407 molecule which has been
previously proposed as NBD1-specific [22]. The difficulty in addressing putative corrector
binding sites is amplified by the fact that some of these molecules may bind conformations
transiently occurring in the early steps of co-translational folding, but no longer existing in
the final native protein like conformation. Of note is that the small size of the SBC molecules
and the flexibility of SBC219 in comparison to the size and rigidity of correctors VX-809, -661,
-
445 and -659 are probably disadvantages to induce an efficient folding and long-term
stability of F508del CFTR. In the search for correctors from the purine scaffold, SBC001 could
be used to introduce large and rigid chemical groups through substitution of the 6-chlorine
atom.
In the field of potentiators, mechanistic studies have proposed that these compounds may
act on the energetic coupling between NBD dimerization and gate opening /closing in the
MSDs [56], shifting gating transitions to favor the open state. This may be performed (i) at
the level of the NBD dimer, by acting on the interface between the NBD1 and NBD2, as
6
targeted by ATP analogues (N -(2-phenyl-ethyl)-ATP (PATP) [57-59], 2'-deoxy-ATP (dATP) [60,
6
6
1] and 2'-deoxy-N -(2-phenyl-ethyl)-adenosine-5'-O-triphosphate (dPATP [62])) or (ii) at the
level of the MSDs, at the interface between the lipid core of the membrane, as proposed for
VX-770 and a more recent potentiator, GLPG1837 [63-66]. Hydrogen/deuterium exchange
studies have indicated some regions particularly affected by VX-770 binding, at the interface
between MSD2 and NBD1 and between MSD2:NBD2 [18], thus distinct from the ATP-binding
and MSD-binding sites. More recently, ivacaftor/ GLPG1837 binding site(s) have been
mapped by docking the compounds on the 3D structures of the CFTR protein [23] and within
an EM density likely corresponding to the drugs within the protein-drug complex [31]. The
mechanisms of action and binding site(s) of a third group of potentiators (5-nitro-2-(3-
phenylpropylamino)benzoate (NPPB)) [67, 68] remain unsettled, as well as those of novel co-
potentiators (arylsulfonamide-pyrrolopyridine ASP11), which activate wild-type CFTR in the
absence of a cAMP agonist [16].
1
4

Here, we have identified potentiator effects induced by a series of yet un-optimized
molecules, which act both, as VX-770, on F508del and wild-type CFTR. The effect of SBCs on
F508del-CFTR does not seem to depend on the type of correction used since it was observed
on both temperature- (screening assays and patch-clamp) and VX-809- (Ussing chamber)
corrected cells and was not affected by the presence of VX-809 during Ussing chamber
experiments. We also show that SBCs act in a synergic manner with VX-770 on several CFTR
mutants (F508del, G551D, G970R and G1349D). There are now several studies emphasizing
the interest of co-potentiators which act in an additive way or allosterically [8, 16, 17, 69-
7
1], some of them being particularly interesting relative to their lack of interference with
corrector activity [8, 17], their nanomolar potency and their effect on VX-770 insensitive
mutations, such as N1303K and W1282X [16, 70]. SBCs remain to be optimized and their
effects to be tested compared to other potentiators (for instance Asp11) and against a large
panel of CFTR pathogenic mutations, especially those which are not addressed by the
recently described class II potentiators, essentially active on mutations in CFTR NBD2 [70].
Even though we observed a negative effect of SBC040 on VX-809 correction of F508del CFTR,
as it is the case for VX-770 and other potentiators [6, 7], SBCs may be of particular interest
for their activity at submaximal concentration of forskolin, as observed for ASP-11 [16],
contrasting with the dependence of VX-770 on the CFTR phosphorylation state [42].
According to Cui and colleagues [42], such drugs acting on sub-maximally activated CFTR are
likely more physiologically relevant than those screened against CFTR channels activated by
high concentrations of PKA activators. Further developing such drugs may enable the
discovery of more potentiators, active on VX-770-resistant mutations, by increasing the
dynamic range and avoiding ceiling effects.
On another hand, SBCs can be considered as tools to understand the mechanisms at play in
CFTR (co-potentiation), in particular relative to our theoretical data, which suggested
potential binding sites at the level of ATP-binding sites, with a noticeable evolution of the
protein conformation to adapt such small molecules and possibly to allow allosteric
regulation of the CFTR channel activity. The relevance of such potentiator binding sites at
the NBDs interface has already been supported by other docking experiments based on 3D
models [72, 73], but remains to be supported at the experimental level. Of note, the free
energy binding values calculated here are lower for SBC219 than for SBC040 in the two
1
5

potential binding sites, a result consistent with (i) the higher efficiency observed for SBC219
and (ii) with the additional contacts predicted to exist between SBC219 and the intracellular
loops (at least at the level of the canonical ATP-binding site). SBC219 is particularly
interesting as it is positioned, at the level of both sites, in between the ATP-binding sites and
ICLs. Based on this theoretical work and its inherent limitations, it is not possible to favor
one site over the other, but the clear differences observed between the binding sites open
perspectives for further characterization and optimization. The canonical binding site seems
particularly appealing as the two NBDs are involved in SBCs binding, thereby enabling the
stabilization of the tight interdomain interface, hallmark of the CFTR open conformation.
Moreover, it also includes, in the specific case of SBC219, amino acid residues from the
intracellular loops (ICLs), thus possibly amplifying allosteric communication with the MSDs
and subsequent channel opening. Maintaining an ATP-bound-like conformation at the
canonical ATP-binding site might prevent closure of the CFTR channel [74]. It is interesting to
note that according to our MD simulations, SBC040 might however still be able to bind in the
vicinity of the canonical ATP-binding site in the G551D-CFTR protein, as the aspartic acid
residue has no direct contact with SBC040 (in contrast to the situation where ATP is bound in
the ATP-binding site [73]), whereas the drug has low or no potentiator effect when used
alone on the mutated protein. This absence of effect may be due to additional bonds that
the aspartic acid might form within the site, which lower the flexibility necessary for
allosteric channel opening at the level of MSDs.
The synergy observed here between VX-770 and SBCs and the theoretical data gained by MD
simulations suggest that obtaining an optimal gating signal might involve distinct sites
present (i) at the level of the NBDs (allowing their optimal association and interaction with
the ICLs) and (ii) at the level of the MSDs (allowing to shift the equilibrium towards an open
conformation of the channel). This synergic (and not only additive) behavior, which was not
observed for other recently developed potentiators ([14-17], might suggest that binding of
one drug molecule to one site may allow allosteric switching towards an intermediate active
conformation, which is then prone to elicit an optimal response to the second drug. Further
studies, involving especially MD simulations with drugs bound in their respective binding
sites, are required to further decipher the molecular mechanisms of allosteric potentiation,
which in turn may inform drug design efforts.
1
6

4. Experimental section
4
.1 SBC synthesis and characterization
All starting materials were commercially available research-grade chemicals and used
without further purification. Reactions were monitored by analytical TLC on silica gel
1
(
Alugram Sil G/UV ) from Macherey–Nagel with fluorescent indicator UV . H NMR
2
54
254
13
spectra were recorded on a Bruker Avance 400 at 400 MHz. C NMR spectra were recorded
either on a Bruker Avance 400 or on a Bruker Avance 500, respectively at 100 or 126 MHz.
Chemical shifts are reported in ppm (parts per million) relative to the residual signal of the
1
solvent in which the spectrum was recorded [ H: δ(d -DMSO) = 2.50 ppm, δ(CDCl ) = 7.27
6
3
1
3
ppm, δ(CD OD) = 3.31 ppm; C: δ(d -DMSO) = 39.52 ppm, δ(CDCl ) = 77.16 ppm, δ(CD OD) =
3
6
3
3
4
9.00 ppm], and the signals are described as singlet (s), broad singlet (bs), doublet (d), triplet
(t), multiplet (m). Coupling constants are reported in Hertz (Hz). Column chromatography
purifications were performed on a Grace Reveleris X1 apparatus, using Grace Resolv silica gel
cartridges (40 µm). HRMS analyses were obtained from the Mass Service, ICOA, at the
University of Orléans, France, using a HRMS Q-Tof MaXis spectrometer. Combustion
analyses were obtained from the analysis facilities of DCM, Grenoble, France. HPLC analyses
were performed with an Agilent 1100 series using a diode array detector and a C reversed-
1
8
phase column (Nucleodur C ISIS, Macherey–Nagel, 5 μm particle size, 250 mm × 4.6 mm),
1
8
with a mobile phase composed of A= H O and TFA 0.1% and B= MeOH and TFA 0.1% with a
2
gradient 100:0 to 0:100 A:B over 20 min, 1 mL/min, 10 μL injection, detection at 254 nm.
Purity of the reported compounds was determined by combustion analysis or HPLC analysis
and in every case appeared to be ≥ 95%. Details of the different syntheses are given in Fig.
S2 (supporting information).
4
.2. SBC activity
4
.2.1. Cell culture
All cell lines were grown at 37°C in 5% CO - 95% air and media were replaced every 2 days.
2
Human bronchial epithelial cell lines were provided by Dr. D. Gruenert (Univ. California San
-
Francisco, USA). CFBE41o cells overexpressing F508del-CFTR (CFBE F508del-CFTR) were
grown on TPP culture flasks in Eagle’s Minimum Essential Medium (EMEM) containing non-
1
7

essential amino acids (NEAA) (Gibco 10370) supplemented with 10% fetal bovine serum
-
1
-1
(
FBS) (Sigma), 2 mM L-glutamine (Gibco), 50 IU.mL penicillin (Sigma), 50 μg.mL of
-
1
streptomycin (Sigma) and were selected using 5 μg.mL puromycin (Gibco). HeLa and HeLa
F508del-CFTR cell lines were grown on TPP culture flasks in Dulbecco's Modified Eagle's
medium containing glutamine and pyruvate (Gibco 31966) supplemented with 9% FBS
-
1
-1
-1
(
Sigma), 50 IU.mL penicillin and 50 μg.mL streptomycin. Zeocin (50 μg.mL , Invitrogen)
was used as selection antibiotic for Hela F508del-CFTR cell line. Hela cells were transiently
transfected with either pEGFP G551D-CFTR or pEGFP G1349D-CFTR cDNA constructs (0.5
-
1
μg.mL ) using cationic lipids (JetPEI, QBiogene) to express G551D-CFTR or G1349D-CFTR
respectively. Cells were used 48h post-transfection. To study the potentiator effect of our
compounds on F508del-CFTR, sufficient levels of the protein were restored to the plasma
membrane by incubating cells at 27°C or with 10 µM VX-809 during 24 h prior experiments.
HEK293 cells purchased from ATCC were cultivated on Falcon culture flasks in Dulbecco's
Modified Eagle's medium containing GlutaMax (Gibco 61965) supplemented with 10% FBS
(
Gibco). HEK293 cells were transiently co-transfected with both halide-sensitive YFP-
H148Q/I152L and pTracer WT-CFTR or mutant CFTR cDNA constructs using cationic lipids
Turbofect, Fermentas). Mutagenesis was performed using the QuickChange XL II
mutagenesis kit (Agilent) following the manufacturer’s instructions using the cDNA of CFTR
M470). Obtained mutants were fully sequenced, amplified, purified (Macherey-Nagel) and
(
(
plasmid concentrations measured using a Nanodrop (Thermo Fisher Scientific). After 24h,
cells were transferred to poly-L-lysine coated 96 well black/clear bottom microplates 96 well
plate.
4
.2.2. Whole-cell patch-clamp recordings
-
For manual patch clamp, Cl currents were measured in the whole-cell patch-clamp
configuration using an Axopatch 200B amplifier connected to an analog/digital interface,
Digidata 1440A, and were analyzed using pCLAMP 9 software (all from Axon Instruments
Inc., Burlingame, CA, USA). The holding potential was -40 mV and voltage pulses from -100
to +100 mV in 20 mV increments were applied to record CFTR currents. Pipettes were pulled
from borosilicate glass capillaries (GC150-TF10; Clark Electromedical Inc., Reading, UK) and
connected to the head stage of the patch clamp amplifier through an Ag–AgCl wire (pipette
resistance 3-4MΩ). Pipette capacitance was electronically compensated in cell-attached
mode. The external bath solution contained (in mM): 145 NaCl, 4 CsCl, 1 CaCl , 1 MgCl , 10
2
2
1
8

glucose, and 10 TES (titrated with NaOH to pH 7.4). The osmolarity was 315 ± 5 mOsmol. The
intrapipette solution contained (in mM): 113 L-aspartic acid, 113 CsOH, 27 CsCl, 1 NaCl, 1
MgCl , 1 EGTA, 1 TES, and 3 Mg-ATP (titrated with CsOH to pH 7.2). The osmolarity was 285
2
±
5 mOsmol.
Automated whole-cell patch clamp (APC) recordings were performed and analyzed as
described previously [75]. Briefly, experiments were performed on the eight-channel
Patchliner NPC-16 workstation (Nanion Technologies GmbH, Munich, Germany), which was
coupled to two QuadroEPC-10 amplifiers (HEKA Elektronik GmbH, Germany). APC
procedures followed Nanion’s standard procedures and used Nanion’s high-resistance chips.
The holding potential was maintained at −40 mV throughout the experiment. To record CFTR
currents, pulses from the holding potential of −40 mV to test potenꢀals between −80 and
+
1
3
1
80 mV in 20 mV increments were used. External solution contained (in mM): 145 NMDG,
45 HCl, 10 TES, 5 BaCl , 2 CaCl , 2 MgCl (titrated with NMDG to pH 7.4). The osmolarity was
2
2
2
00 ± 10 mOsmol. Internal solution contained (in mM): 105 NMDG, 30 H SO , 20 HCl, 10 TES,
2
4
0 EGTA, 4 MgCl , 3 Mg-ATP (titrated with HCl to pH 7.2). The osmolarity was 285 ± 5
2
mOsmol. All patch clamp experiments (manual and automatic) were conducted at room
temperature (20 –25 °C).
4
.2.3. Short-circuit current measurements
6
CFBE CFTR-F508del cells were seeded at a density of 0.5x10 cells on Snapwell permeable
-
2
inserts (#3407, Corning Corp.) coated with human fibronectin (5 μg.cm ; Sigma). After 2
days at liquid/liquid interface, cells were cultured at air/liquid interface until transepithelial
-
2
resistance reached a minimum of 300 Ω.cm (5-6 days). Transepithelial resistances were
measured with a Millicell-ERS voltmeter-ohmmeter (Millipore). Inserts containing pseudo-
epithelia were then mounted in an EM-CSYS-6 Ussing chamber system (Physiologic
Instruments Inc., USA) composed of two hemi-chambers, each containing a different
-
solution. Asymmetric solutions were used, creating a basal to apical Cl gradient to enhance
-
Cl currents detection. Their composition was (in mM): 1.2 NaCl, 115 Na-Gluconate, 25
NaHCO , 1.2 MgCl , 4 CaCl , 2.4 KH PO , 1.24 K HPO , 10 mannitol (pH 7.4) for apical
3
2
2
2
4
2
4
solution and 115 NaCl, 25 NaHCO , 1.2 MgCl , 1.2 CaCl , 2.4 KH PO , 1.24 K HPO , 10 glucose
3
2
2
2
4
2
4
(pH 7.4) for basal solution. Apical and basal solutions were maintained around physiological
temperature (35-38°C) and gassed with 95% O - 5% CO . Transepithelial potential difference
2
2
and short-circuit currents were measured/injected through 3M KCl filled Ag/AgCl electrodes
1
9

connected to a VCC MC2 voltage/current clamp (Physiologic Instruments Inc., USA).
Visualization and recording of the current injected by the system to short-circuit pseudo-
epithelia (clamp at 0 mV) was visualized and recorded at a frequency of 0.1 Hz on a personal
computer using Acquire and Analyze hardware and software (Physiologic Instruments Inc.,
USA). Transepithelial potential difference values were corrected for the junction potential
between apical and basal solutions and for empty insert resistance. Since the polarity of Isc
was referred to the basal side of the pseudo-epithelium and a gain of -10 was applied, an
apical anion secretion was indicated by an increase in Isc. All experiments were done in the
presence of 100 µM amiloride in the apical solution to prevent ENaC currents. All the drugs
were added to the apical solution.
4
.2.4. Intracellular cAMP measurements
CFBE CFTR-F508del cells were plated in 60 mm diameter culture dishes (400,000 cells per
dish). When cells reached 80% confluence, they were treated with test compounds at the
concentration leading to the maximal potentiator effect during 15 min at 37°C. Intracellular
contents were harvested by incubating cells during 20 min in a 0.1M HCl solution, followed
by a centrifugation step at 18000 x g. Intracellular cAMP levels were then measured using a
cAMP Direct Immunoassay kit (ab65355, Abcam), according to the kit manufacturer
instructions. Data are expressed as the percentage of intracellular cAMP levels detected in
solvent treated cells.
4
.2.5. Cell viability test:
Hela cells were seeded in 96 well plates (30,000 cells per well). 24h later, cells were
incubated at 37°C with test compounds at various concentrations during 24h. Wells were
then washed with Phosphate Buffer Saline (PBS) and incubated with a solution of Thiazolyl
-
1
Blue Tetrazolium Bromide (MTT) (0.5 mg.mL ; Sigma) during 4h before the supernatant was
removed. Purple formazan crystals were dissolved in 100 µL dimethylsulfoxide (Sigma) and
optical density was measured at 570 nm with a 630 nm correction using a microplate reader
(
SpectraCount microplate photometer; Packard, Meriden, CT). Data are expressed as the
percentage of values obtained for solvent treated cells (DMSO).
.2.6. Western-blot
4
Hela cells were lysed (lysis buffer: 10 mM Tris HCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 1 mM de Pefabloc® SC and protease inhibitors cocktail; pH 7.5). 50 µg of
protein were resolved on a 5-10% gradient SDS-PAGE, transferred to a nitrocellulose
2
0

membrane, immunoblotted using the MAB3480 anti-CFTR antibody and the Na/K ATPase
both mouse monoclonal used at 1:1000; Millipore Corporation, USA), exposed to secondary
(
antibody conjugated to horseradish peroxidase (used at 1:5000; Amersham, GE Healthcare,
UK) for 1 hour and revealed by chemiluminescence with ECL Western-blotting reagent
(Millipore) and the GeneGnome XRQ chemiluminescence systems (Syngen, Synoptic Ltd,
UK).
4
.2.7. YFP measurements
After 24 hours, transfected HEK293 cells in 96-well plates were washed with PBS and each
well incubated 30 min with 50 µL of PBS containing cpt-cAMP (100µM) and IBMX (100 µM)
(
all from Sigma-Aldrich) with and without VX-770, SBC040 or SBC219. Plates were then
transferred to a ClarioStar plate-reader (BMG) to measure YFP fluorescence. After 3 seconds,
00 µl of PBS-NaI (PBS solution where NaCl is replaced with NaI) were injected. Signal decay
2
was fitted to an exponential function to derive the maximal slope corresponding to initial
-
influx into the cells. Maximal slopes were converted to rates of change in intracellular I
concentration (in mM/s).
4
.2.8. Drugs and chemicals
Amiloride, CFTR(inh)-172, forskolin, genistein, cpt-cAMP and IBMX were purchased from
Sigma Aldrich (France). VX-770 and VX-809 were obtained from Selleckchem (USA). Stock
solutions of SBCs and other drugs were prepared using dimethyl sulfoxide (DMSO) as
solvent.
4
.2.9. Data analysis and statistics:
For patch-clamp and Ussing chamber experiments, graphs and statistics were done using
Prism 5 (GraphPad Software, USA) and all the results are expressed as mean ± SEM except
for box plot representations for which the central line represents the median, boxes upper
th
th
and lower edges represent the 25 and 75 percentile respectively while the whiskers
represent the minimal and maximal values of the sample. Statistical comparisons were made
using non-parametric (n < 10) or parametric (n ≥ 10) tests with a significance level of 0.05.
Before using a parametric test, samples were checked for normality using Shapiro-Wilk
normality test. For Ussing chamber screening data, since these experiments did not require
high replication level, a parametric test was used even if the sample size was small and
normality of sample distribution could not be tested. For YFP based measurements,
quantitative variables are described as mean (± SEM). Comparisons to WT conditions and
2
1

between treated and untreated conditions were made with one-way ANOVA followed by
Fischer test for p evaluation.
4
.3. Docking and molecular dynamics simulations
4
.3.1. CFTR models
We used the published model of the open form of the CFTR MSD:NBD assembly [25] which
was supported by experimental data [44] including recent cryo-electron structures of the
full-length CFTR [30]. Based on the wild-type (WT) model, we built using chimera [76, 77]
three additional models including mutations. The first corresponds to the most common
class II mutant, a CFTR-F508del model while the other models represent class III G551D and
G1349D mutants.
4
.3.2. Design of SBCs and docking on CFTR
SBC040 and SBC219 were designed using the Ligand Reader & Modeler module of the
CHARMM-GUI platform [78-80]t. CHARMM-compatible topology and parameter files were
hence generated using the CgenFF tool [81-83]. The ligands were docked either within CFTR
canonical or non-canonical sites after a superimposition with the ATP occupying each site.
These manual docking was also supported by a docking performed using Autodock Vina [84]
and AutoDockTools from MGLTools package with ATP-free CFTR. Using a distance cutoff of
1
2 Å between the centers of mass of ATP and each SBC position, we found that 45% of
positions out of the from the top twenty docking hits were within ATP-binding sites for both
SBC040 and SBC219 (by comparison, we found 60 % (SBC040) and 65% (SBC219) matching
positions in the cryo-EM hCFTR structure).
4
.3.3. Molecular dynamics simulations
Each model was embedded in a lipid bilayer consisting of POPC molecules using the
charmm-gui platform [78, 80, 85, 86] and the same protocol as previously described [44] for
generating and equilibrating protein-membrane simulations complexes, as well as for the
MD simulations (CHARM36 force field [87] with the CMAP correction [88] in NAMD 2.9 [89]).
Production phases were conducted (the simulation time of each system is reported in Figure
S7 (supporting information)) at a temperature of 310 K (wild-type CFTR, CFTR-G551D and
CFTR-G1349D) or 300 K (for CFTR-F508del) and a pressure of 1 bar.
2
2

4
.3.4. Contact analysis
The contacts between each SBC and CFTR were analyzed by VLDM (Voronoi Laguerre
Delaunay for Macromolecules) [90-92]. VLDM relies on a tessellation method, that is, a
partition of space into a collection of polyhedra filling space without overlaps or gaps. The
program builds the Delaunay tessellation and its Laguerre dual from a set of atomic data,
each atom being characterized by its position in space and a weight depending on its van der
Waals radius. For consistency with the simulations, the van der Waals radius values are
those of CHARMM36 [87]. In the present analysis, only the heavy atoms of the protein,
membrane and solvent were considered. CFTR/SBCs contact maps were realized using R
[
93].
.3.5. Cluster analysis
4
SBCs binding sites were precisely defined by these contact map analyses. Using only these
protein sites definition, clustering analyses were performed using the contact map overlap
(CMO) as a metric in ClusCo program [94].
4
.3.6. Additional Trajectory Analyses
Root Mean Square Deviations (RMSDs) were computed with VMD between the snapshots
and the initial configuration derived from CFTR model before the heating and equilibration
stages. RMSDs were calculated on protein backbone heavy atoms. Salt bridges and atom
pairs distances were calculated using in-house tcl scripts executed in VMD. Figures of RMSDs
were prepared using gnu plot 5.0 (http://www.gnuplot.info.).
MD trajectories were visualized with VMD. Snapshots figures were made by CHIMERA [77],
Pymol [95] or VMD [96].
4
.3.7. Binding Free Energy calculations
We performed Molecular Mechanics/ Generalized Born Surface Area (MMGBSA) calculations
using the python script MMPBSA.py (in Ambertools 18) [97] to provide an estimation and a
comparison of the binding free energies of the SBCs in their different binding sites. This tool
is a post-processing method in which representative snapshots from MD trajectories are
used to calculate the free energy change between SBC bound and free state of CFTR. We
used default parameters with igb= 2.
A
CKNOWLEDGMENTS
2
3

This work was performed using HPC resources from GENCI-[CINES] (Grants 2017-
A0020707206, 2018-A0040707206, 2019-A006707206) and benefited from the financial
support of the French Association Vaincre la Mucoviscidose (VLM) and Association pour
l’Aide à la Recherche contre la Mucoviscidose (AARM). It was also partially supported by
Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003). A.B. was the recipient of post-
doctoral fellowship from the CF Trust.
R
EFERENCES
[
[
1] J.S. Elborn, Cystic fibrosis, Lancet, 388 (2016) 2519-2531.
2] L. Csanády, P. Vergani, D.C. Gadsby, Structure, gating, and regulation of the CFTR anion
channel., Physiol Rev, 99 (2019) 707-738.
3] G. Veit, R.G. Avramescu, A.N. Chiang, S.A. Houck, Z. Cai, K.W. Peters, J.S. Hong, H.B.
[
Pollard, W.B. Guggino, W.E. Balch, W.R. Skach, G.R. Cutting, R.A. Frizzell, D.N. Sheppard,
D.M. Cyr, E.J. Sorscher, J.L. Brodsky, G.L. Lukacs, From CFTR biology toward combinatorial
pharmacotherapy: expanded classification of cystic fibrosis mutations., Mol Biol Cell, 27
(2016) 424-433.
[
4] F. Van Goor, S. Hadida, P.D. Grootenhuis, B. Burton, J.H. Stack, K.S. Straley, C.J. Decker,
M. Miller, J. McCartney, E.R. Olson, J.J. Wine, R.A. Frizzell, M. Ashlock, P.A. Negulescu,
Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug
VX-809., Proc Natl Acad Sci U S A, 108 (2011) 18843-18848.
[
5] C.E. Wainwright, J.S. Elborn, B.W. Ramsey, G. Marigowda, X. Huang, M. Cipolli, C.
Colombo, J.C. Davies, K. De Boeck, P.A. Flume, M.W. Konstan, S.A. McColley, K. McCoy, E.F.
McKone, A. Munck, F. Ratjen, S.M. Rowe, D. Waltz, M.P. Boyle, T.S. Group, T.S. Group,
Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR., N
Engl J Med, 373 (2015) 220-231.
[
6] D.M. Cholon, N.L. Quinney, M.L. Fulcher , C.R.J. Esther, J. Das, N.V. Dokholyan, S.H.
Randell, R.C. Boucher, M. Gentzsch, Potentiator ivacaftor abrogates pharmacological
correction of DeltaF508 CFTR in cystic fibrosis. , Sci Transl Med, 6 (2014) 246ra296.
[
7] G. Veit, R.G. Avramescu, D. Perdomo, P.W. Phuan, M. Bagdany, P.M. Apaja, F. Borot, D.
Szollosi, Y.S. Wu, W.E. Finkbeiner, T. Hegedus, A.S. Verkman, G.L. Lukacs, Some gating
potentiators, including VX-770, diminish DeltaF508-CFTR functional expression. , Sci Transl
Med, 6 (2014) 246ra297.
[
8] P.W. Phuan, G. Veit, J. Tan, A. Roldan, W.E. Finkbeiner, G.L. Lukacs, A.S. Verkman,
Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-
CFTR correctors that augment VX-809 maximal efficacy., Mol Pharmacol, 86 (2014) 42-51.
[
9] C. Boinot, M. Jollivet Souchet , R. Ferru-Clément, F. Becq, Searching for combinations of
small-molecule correctors to restore f508del-cystic fibrosis transmembrane conductance
regulator function and processing., J Pharmacol Exp Ther, 350 (2014) 624-634.
[
10] G. Veit, H. Xu, E. Dreano, R.G. Avramescu, M. Bagdany, L.K. Beitel, A. Roldan, M.A.
Hancock , C. Lay, W. Li, K. Morin, S. Gao, P.A. Mak, E. Ainscow, A.P. Orth, P. McNamara, A.
Edelman, S. Frenkiel, E. Matouk, I. Sermet-Gaudelus, W.G. Barnes, G.L. Lukacs Structure-
guided combination therapy to potently improve the function of mutant CFTRs., Nat Med, 24
(2018) 1732-1742.
2
4

[
11] G.W. Carlile, Q. Yang, E. Matthes, J. Liao, S. Radinovic, C. Miyamoto, R. Robert, J.W.
Hanrahan, D.Y. Thomas, A novel triple combination of pharmacological chaperones improves
F508del-CFTR correction., Sci Rep, 8 (2018) 11404.
[
12] J. Davies, S.M. Moskowitz, C. Brown, A. Horsley, M.A. Mall, E.F. McKone, B.J. Plant, D.
Prais, B.W. Ramsey, J.L. Taylor-Cousar, E. Tullis, A. Uluer, C.M. McKee, S. Robertson, R.A.
Shilling, C. Simard, F. Van Goor, D. Waltz, F. Xuan, T. Young, S.M. Rowe, V.-.-S. Group., VX-
6
59-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles.,
N Engl J Med, 379 (2018) 1599-1611.
13] D. Keating, G. Marigowda, L. Burr, C. Daines, M.A. Mall, E.F. McKone, B.W. Ramsey, S.M.
[
Rowe, L.A. Sass, E. Tullis, C.M. McKee, S.M. Moskowitz, S. Robertson, J. Savage, C. Simard, F.
Van Goor, D. Waltz, F. Xuan, T. Young, J.L. Taylor-Cousar, V.-.-S. Group., VX-445-Tezacaftor-
Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles., N Engl J Med,
3
79 (2018) 1612-1620.
[
14] M. Gees, S. Musch, S. Van der Plas, A.S. Wesse, A. Vandevelde, K. Verdonck, O.
Mammoliti, T. Hwang, K. Sonck, P. Stouten, A.M. Swensen, M. Jans, J. Van der Schueren, L.
Nelles, M. Andrews, K. Conrath, Identification and Characterization of novel CFTR
potentiators., Front Pharmacol, 9 (2018) 221.
[
15] J. Park , P. Khloya, Y. Seo, S. Kumar, H.K. Lee, D.K. Jeon, S. Jo, P.K. Sharma, W. Namkung,
Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines.,
PLoS One, 11 (2016) e0149131.
[
16] P.W. Phuan, J.H. Son, J.A. Tan, C. Li, I. Musante, L. Zlock, D.W. Nielson, W.E. Finkbeiner,
M.J. Kurth, L.J. Galietta, P.M. Haggie, A.S. Verkman, Combination potentiator ('co-
potentiator') therapy for CF caused by CFTR mutants, including N1303K, that are poorly
responsive to single potentiators., J Cyst Fibr, 17 (2018) 595-606.
[
17] P.W. Phuan, G. Veit, J.A. Tan, W.E. Finkbeiner, G.L. Lukacs, A.S. Verkman, Potentiators of
Defective ΔF508-CFTR Gating that Do Not Interfere with Corrector Action., Mol Pharmacol,
8 (2015) 791-799.
18] L. Byrnes, Y. Xu, X. Qiu, J. Hall, G. West, Sites associated with Kalydeco binding on
8
[
human Cystic Fibrosis Transmembrane Conductance Regulator revealed by
Hydrogen/Deuterium Exchange., Sci Rep, 8 (2018) 4664.
[
19] I. Callebaut, B. Hoffmann, J.-P. Mornon, The implications of CFTR structural studies for
cystic fibrosis drug development., Curr Opin Pharmacol, 34 (2017) 112-118.
20] O. Kalid, M. Mense, S. Fischman, A. Shitrit, H. Bihler, E. Ben-Zeev, N. Schutz, N.
[
Pedemonte, P.J. Thomas, R.J. Bridges, D.R. Wetmore, Y. Marantz, H. Senderowitz, Small
molecule correctors of F508del-CFTR discovered by structure-based virtual screening., J
Comput Aided Mol Des, 24 (2010 ) 971-991.
[
21] S.V. Molinski, V.M. Shahani, A.S. Subramanian, S.S. MacKinnon, G. Woollard, M. Laforet,
O. Laselva, L.D. Morayniss, C.E. Bear, A. Windemuth, Comprehensive mapping of cystic
fibrosis mutations to CFTR protein identifies mutation clusters and molecular docking
predicts corrector binding site., Proteins, 86 (2018) 833-843.
[
22] N. Odolczyk, J. Fritsch, C. Norez, N. Servel, M.F. da Cunha, S. Bitam, A. Kupniewska, L.
Wiszniewski, J. Colas, K. Tarnowski, D. Tondelier, A. Roldan, E.L. Saussereau, P. Melin-
Heschel, G. Wieczorek, G.L. Lukacs, M. Dadlez, G. Faure, H. Herrmann, M. Ollero, F. Becq, P.
Zielenkiewicz, A. Edelman, Discovery of novel potent ΔF508-CFTR correctors that target the
nucleotide binding domain., EMBO Mol Med, 5 (2013) 1484-1501.
[
23] H.-I. Yeh, L. Qiu, Y. Sohma, K. Conrath, X. Zou, T. Hwang, Identifying the molecular target
sites for CFTR potentiators GLPG1837 and VX-770., J Gen Physiol, 151 (2019) 912-928.
2
5

[
24] Z. Zhang, J. Chen, Atomic Structure of the Cystic Fibrosis Transmembrane Conductance
Regulator., Cell, 167 (2016) 1586-1597.
25] J.-P. Mornon, B. Hoffmann, S. Jonic, P. Lehn, I. Callebaut, Full-open and closed CFTR
[
channels, with lateral tunnels from the cytoplasm and an alternative position of the F508
region, as revealed by molecular dynamics., Cell Mol Life Sci, 72 (2015) 1377-1403.
[
26] J.-P. Mornon, P. Lehn, I. Callebaut, Atomic model of human cystic fibrosis
transmembrane conductance regulator: membrane-spanning domains and coupling
interfaces., Cell Mol Life Sci, 65 (2008) 2594-2612.
[
27] A.W. Serohijos, T. Hegedus, A.A. Aleksandrov, L. He, L. Cui, N.V. Dokholyan, J.R. Riordan,
Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D
structure crucial to assembly and channel function., Proc Natl Acad Sci U S A, 105 (2008)
3
256-3261.
28] F. Liu, Z. Zhang, L. Csanády, D.C. Gadsby, J. Chen, Molecular structure of the human
CFTR ion channel., Cell, 169 (2017) 85-95.
29] Z. Zhang, F. Liu, J. Chen, Conformational changes of CFTR upon phosphorylation and ATP
binding., Cell, 170 (2017) 483-491.
30] Z. Zhang, F. Liu, J. Chen, Molecular structure of the ATP-bound, phosphorylated human
CFTR., Proc Natl Acad Sci U S A, 115 (2018) 12757-12762.
31] F. Liu, Z. Zhang, A. Levit, J. Levring, K.K. Touhara, B.K. Shoichet, J. Chen, Structural
identification of a hotspot on CFTR for potentiation., Science, 364 (2019) 1184-1188.
32] S. Bach, M. Knockaert, J. Reinhardt, O. Lozach, S. Schmitt, B. Baratte, M. Koken, S.
[
[
[
[
[
Coburn, L. Tang, T. Jiang, D. Liang, H. Galons, J. Dierick, L. Pinna, F. Meggio, F. Totzke, C.
Schachtele, A. Lerman, A. Carnero, Y. Wan, N. Gray, L. Meijer, Roscovitine targets, protein
kinases and pyridoxal kinase. , J Biol Chem, 280 (2005) 31208-31219.
[
33] M.E. Welsch, S.A. Snyder, B.R. Stockwell, Privileged Scaffolds for Library Design and
Drug Discovery Curr. Opin. Chem. Biol, 14 (2010) 347–361.
34] S. Dinesh, G. Shikha, G. Bhavana, S. Nidhi, S. Dileep, Biological activities of purine
analogues and scientific innovation, J Pharm Sci Innov, 1 (2012) 29-34.
35] P.Y. Lam, C.G. Clark, S. Saubern, J. Adams, M.P. Winters, D.M. Chan, A. Combs, New N-
and O-arylations with phenylboronic acids and cupric acetate., Tetrahedron Lett, 39 (1998)
941–2944.
36] L.A. Foller, T. Ulven, Direct N9-arylation of purines with aryl halides. , Chem. Commun,
0 (2014) 4997-4999.
37] M. Dejmek, S. Kovacokova, E. Zbornikova, H. Hrebabecky, M. Sala, M. Dracinsky, R.
[
[
2
[
5
[
Nencka, One-pot build-up procedure for the synthesis of variously substituted purine
derivatives., RSC Advances, 2 (2012) 6970–6980.
[
38] J.-L. Décout, R. Zelli, W. Zeinyeh, B. Boucherle, R. Haudecoeur, Methods for synthesizing
diversely substituted purines. PCT Int. Appl. (2018), , in: U.G. Alpes (Ed.), 2018.
39] R. Zelli, W. Zeinyeh, J.-L. Décout, 6-Chloropurine ribonucleosides from
chloropyrimidines: One pot synthesis. , Current Protocols in Nucleic Acid Chemistry, e57
[
(2018) 1-10.
[
40] R. Zelli, W. Zeinyeh, R. Haudecoeur, J. Alliot, B. Boucherle, I. Callebaut, J.-L. Décout, A
one-pot synthesis of highly substituted purines., Org. Lett., 19 (2017) 6300–6363.
41] F. Van Goor, S. Hadida, P.D. Grootenhuis, B. Burton, D. Cao, T. Neuberger, A. Turnbull, A.
[
Singh, J. Joubran, A. Hazlewood, J. Zhou, J. McCartney, V. Arumugam, C. Decker, J. Yang, C.
Young, E.R. Olson, J.J. Wine, R.A. Frizzell, M. Ashlock, P. Negulescu, Rescue of CF airway
2
6

epithelial cell function in vitro by a CFTR potentiator, VX-770., Proc Natl Acad Sci U S A, 106
(2009) 18825-18830.
[
42] G. Cui, B.B. Stauffer, B.R. Imhoff, R. A, H.J. S, E.J. Sorscher, N.A. McCarty, VX-770-
mediated potentiation of numerous human CFTR disease mutants is influenced by
phosphorylation level., Sci Rep, 9 (2019) 13460.
[
43] H. Yu, B. Burton, C.J. Huang, J. Worley, D. Cao, J.P.J. Johnson, A. Urrutia, J. Joubran, S.
Seepersaud, K. Sussky, B.J. Hoffman, F. Van Goor, Ivacaftor potentiation of multiple CFTR
channels with gating mutations., J Cyst Fibros, 11 (2012) 237-245.
[
44] B. Hoffmann, A. Elbahnsi, P. Lehn, J.-L. Décout, F. Pietrucci, J.-P. Mornon, I. Callebaut,
Combining theoretical and experimental data to decipher CFTR 3D structures and functions.,
Cell Mol Life Sci 75 (2018) 3829-3855.
[
45] A. Billet, J.-P. Mornon, M. Jollivet, P. Lehn, I. Callebaut, F. Becq, CFTR: Effect of ICL2 and
ICL4 amino acids in close spatial proximity on the current properties of the channel., J Cyst
Fibros, 12 (2013) 737-745.
[
46] W. Wang, B.C. Roessler, K.L. Kirk, An electrostatic interaction at the tetrahelix bundle
promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator
(CFTR) channel opening., J Biol Chem, 289 (2014) 30364-30378.
[
47] G. Cui, C.S. Freeman, T. Knotts, C.Z. Prince, C. Kuang, N.A. McCarty, Two salts bridges
differentially contribute to the maintenance of Cystic Fibrosis Transmebrane Conductance
Regulator (CFTR) channel function., J Biol Chem, 288 (2013) 20758-20767.
[
48] G. Cui, Z.R. Zhang, A.R. O'Brien, B. Song, N.A. McCarty, Mutation at arginine 352 alters
the pore architecture of CFTR., J Membr Biol., 222 (2008) 91-106.
49] O. Laselva, S. Molinski, V. Casavola, C.E. Bear, Correctors of the Major Cystic Fibrosis
Mutant Interact through Membrane-Spanning Domains., Mol Pharmacol, 93 (2018) 612-618.
50] H.Y. Ren, D.E. Grove, S.A. Houck, P. Sopha, F. van Goor, B.J. Hoffman, D.M. Cyr, VX-809
[
[
corrects folding defects in cystic fibrosis transmembrane conductance regulator protein
through action on membrane-spanning domain 1., Mol Biol Cell, 24 (2013) 3016-3024.
[
51] R.P. Hudson, J.E. Dawson, P.A. Chong, Z. Yang, L. Millen, P.J. Thomas, C.G. Brouillette,
J.D. Forman-Kay, Direct binding of the corrector VX-809 to human CFTR NBD1: Evidence of
an allosteric coupling between the binding site and the NBD1:CL4 interface., Mol Pharmacol,
9
2 (2017) 124-135.
[
52] W.M. Rabeh, F. Bossard, H. Xu, T. Okiyoneda, M. Bagdany, C.M. Mulvihill, K. Du, S. di
Bernardo, Y. Liu, L. Konermann, A. Roldan, G.L. Lukacs, Correction of both NBD1 energetics
and domain interface is required to restore ΔF508 CFTR folding and function, Cell, 148
(2012) 150-163.
[
53] M. Sigoillot, M. Overtus, M. Grodecka, D. Scholl, A. Garcia-Pino , T. Laeremans, L. He, E.
Pardon, E. Hildebrandt, I. Urbatsch, J. Steyaert, J.R. Riordan, C. Govaerts, Domain-interface
dynamics of CFTR revealed by stabilizing nanobodies., Nat Commun, 10 (2019) 2636.
[
54] L. He, A.A. Aleksandrov, J. An, L. Cui, Z. Yang, C.G. Brouillette, J.R. Riordan, Restoration
of NBD1 thermal stability is necessary and sufficient to correct DF508 CFTR folding and
assembly., J Mol Biol, 427 (2015) 106-120.
[
55] J.D. Hall, H. Wang, L.J. Byrnes, S. Shanker, K. Wang, I.V. Efremov, P.A. Chong, J.D.
Forman-Kay, A.E. Aulabaugh, Binding screen for cystic fibrosis transmembrane conductance
regulator correctors finds new chemical matter and yields insights into cystic fibrosis
therapeutic strategy., Protein Sci, 25 (2016) 360-373.
[
56] K.Y. Jih, T.C. Hwang, Nonequilibrium gating of CFTR on an equilibrium theme.,
Physiology, 27 (2012) 351-361.
2
7

[
57] S.G. Bompadre, M. Li, T.C. Hwang, Mechanism of G551D-CFTR (cystic fibrosis
transmembrane conductance regulator) potentiation by a high affinity ATP analog., J Biol
Chem, 283 (2008) 5364-5369.
[
58] M.F. Tsai, K.Y. Jih, H. Shimizu, M. Li , T.C. Hwang, Optimization of the degenerated
interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis
transmembrane conductance regulator (CFTR) channels., J Biol Chem, 285 (2010) 37663-
3
[
7671.
59] Z. Zhou, X. Wang, M. Li, Y. Sohma, X. Zou, T.C. Hwang, High affinity ATP/ADP analogues
as new tools for studying CFTR gating., J Physiol. , 569 (2005) 447-457.
60] A.A. Aleksandrov, L. Aleksandrov, J.R. Riordan, Nucleoside triphosphate pentose ring
impact on CFTR gating and hydrolysis., FEBS Lett, 518 (2002) 183-188.
61] Z. Cai, A. Taddei, D.N. Sheppard, Differential sensitivity of the cystic fibrosis (CF)-
[
[
associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane
conductance regulator (CFTR) Cl- channel., J Biol Chem., 281 (2006) 1970-1977.
[
62] H. Miki, Z. Zhou, M. Li, T.C. Hwang, S.G. Bompadre, Potentiation of disease-associated
cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.,
J Biol Chem, 285 (2010) 19967-19975.
[
63] P.D. Eckford, C. Li, M. Ramjeesingh, C.E. Bear, Cystic fibrosis transmembrane
conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel
gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner., J Biol
Chem, 287 (2012) 36639-36649.
[
64] K.Y. Jih, T.C. Hwang, Vx-770 potentiates CFTR function by promoting decoupling
between the gating cycle and ATP hydrolysis cycle., Proc Natl Acad Sci U S A, 110 (2013)
404-4409
65] H. Yeh, Y. Sohma, K. Conrath, T. Hwang, A common mechanism for CFTR potentiators., J
Gen Physiol, 149 (2017) 1105-1118.
66] H.I. Yeh, J.T. Yeh, T.C. Hwang, Modulation of CFTR gating by permeant ions., J Gen
Physiol, 145 (2015) 47-60.
67] W.-Y. Lin, Y. Shoma, T.C. Hwang, Synergistic Potentiation of Cystic Fibrosis
4
[
[
[
Transmembrane Conductance Regulator Gating by Two Chemically Distinct Potentiators,
Ivacaftor (VX-770) and 5-Nitro-2-(3-Phenylpropylamino) Benzoate, Mol Pharmac, 90 (2016)
2
[
75-286.
68] W. Wang, G. Li, J.P. Clancy, K.L. Kirk, Activating cystic fibrosis transmembrane
conductance regulator channels with pore blocker analogs., J Biol Chem, 280 (2005) 23622-
3630.
69] Caputo, A, A. Hinzpeter, E. Caci, N. Pedemonte, N. Arous, M. Di Duca, O. Zegarra-Moran,
2
[
P. Fanen, L.J. Galietta, Mutation-specific potency and efficacy of cystic fibrosis
transmembrane conductance regulator chloride channel potentiators., J Pharmacol Exp
Ther, 330 (2009) 783-791.
[
70] P.W. Phuan, J.A. Tan, A.A. Rivera, L. Zlock, D.W. Nielson, W.E. Finkbeiner, P.M. Haggie,
A.S. Verkman, Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a
defined subset of minimal function CFTR mutants., Sci Rep, 27 (2019) 17640.
[
71] G. Veit, D.F. Da Fonte, R.G. Avramescu, A. Premchandar, M. Bagdany, H. Xu, D.
Bensinger, D. Stubba, B. Schmidt, E. Matouk, G.L. Lukacs, Mutation-specific dual potentiators
maximize rescue of CFTR gating mutants, J Cyst Fibr, in press (2019).
2
8