INTRODUCTION
Cystic fibrosis (CF) is the most common life-threating autosomal
recessive disease among Caucasians, affecting almost 50,000 individuals
in Europe (ECFS Patient Registry, 2017). It is caused by mutations in
the gene encoding the CF transmembrane conductance regulator (CFTR)
protein, which functions as a chloride (Cl-) and
bicarbonate (HCO3-) channel at the
apical plasma membrane (PM) of epithelial cells (Riordan, 2008).
CF-causing mutations cause channel dysfunction, leading to abnormal ion
transport and dehydration of epithelia in several tissues (De Boeck &
Amaral, 2016; Lopes-Pacheco, 2016). Although CF is a multi-organ
disease, the respiratory disorder represents the major cause of
morbidity and mortality of individuals with CF due to airway obstruction
by a thick mucus, chronic inflammation and persistent infections, which
ultimately result in respiratory failure (De Boeck & Amaral, 2016;
Lopes-Pacheco, 2016).
CFTR protein is composed of two transmembrane domains (TMD1/2), two
nucleotide-binding domains (NBD1/2) and a regulatory domain (RD). The
TMDs form the pore through which anions are conducted along their
electrochemical gradient, while the NBDs regulate channel gating by
binding and hydrolyzing ATP and after RD phosphorylation at multiple
sites. Interdomain interactions are critical for this complex protein to
achieve its native conformation state (Riordan, 2008).
Over 2,000 CFTR gene variants have been reported so far
(http://www.genet.sickkids.on.ca/), with deletion of a
phenylalanine at position 508 (F508del in NBD1) being the most prevalent
and occurring in ~80% of individuals with CF in Europe,
albeit with some geographic variability (ECFS Patient Registry, 2017).
F508del causes CFTR protein misfolding that is recognized by the
endoplasmic reticulum (ER) quality control (ERQC) machinery and
prematurely degraded by the proteasome (Jensen et al., 1995).
Rescue of F508del-CFTR was first demonstrated by low temperature
incubation of cells heterologously expressing this mutant (Denninget al., 1992), thus proving that this mutant is both temperature
sensitive and rescuable.
Over the past decade, significant efforts have been put into
high-throughput screening (HTS) of small molecule libraries to identify
compounds that rescue the F508del-CFTR protein to the PM. To date, there
are three correctors approved for clinical use by the Food and Drug
Administration (FDA), being two also approved by the European Medicine
Agency (EMA) (all combined with potentiator VX-770/ivacaftor):
VX-809/lumacaftor, VX-661/tezacaftor and VX-445/elexacaftor (only
FDA-approved). In clinical trials, individuals who were
F508del-homozygous and treated with either VX-809 or VX-661 plus VX-770
demonstrated a significant, albeit modest, improvement in lung function
(Wainwright et al., 2015; Taylor-Cousar et al. 2017). More
recently, VX-445 was added to the co-treatment with VX-661/VX-770 and
this triple combination demonstrated greater therapeutic benefit in
phase 3 clinical trials (Heijerman et al., 2019; Middletonet al., 2019), thus leading to its FDA-approval in individuals
with CF, aged ≥12 years and with the F508del mutation in at least one
allele.
Despite such progress, individuals with CF still face several
disease-related symptoms and complications, including a progressive
deterioration of lung function, and thus novel correctors are still
needed to achieve more potent combinations. Furthermore, there are other
CFTR trafficking mutants that are not efficiently rescued by available
correctors, including G85E and N1303K (Dekkers et al., 2016;
Lopes-Pacheco et al., 2017). Along these lines, the novel
RDR01752 compound was identified as a F508del-CFTR traffic corrector in
a small-scale screen (Carlile et al., 2007) and demonstrated to
thermally stabilize purified murine F508del-NBD1 in vitro(Sampson et al., 2011). However, its mechanism of action (MoA)
remains to be elucidated.
Here, we investigated the MoA of RDR01752 in cell lines stably
expressing either F508del-CFTR or other CFTR mutants, and in
F508del/F508del intestinal organoids. The MoA of RDR01752 was explored
by analyzing its additive effects to those of previously described CFTR
genetic revertants. These are second-site mutations, i.e., in ciswith F508del that partially rescue F508del-CFTR. One of these revertants
results from removal of the arginine-framed motifs (AFT) acting as
retention signals (4RK), thus allowing the mutant protein to escape the
ERQC (Chang et al., 1999; Roxo-Rosa et al., 2006; Farinhaet al., 2013). Two others work by correcting folding at critical
structural pockets present in the 3D-structure of F508del-CFTR that are
absent in wild-type (WT)-CFTR. These include G550E that acts by
stabilizing the NBD1:NBD2 dimer interface (Roxo-Rosa et al.,2006) and R1070W that restores the NBD1:ICL4 interaction (Serohijoset al., 2008; Thibodeau et al., 2010; Farinha et
al., 2013). We also investigated the effects of RDR01752 on the DD/AA
variant on the background of WT-CFTR, which lacks the double diacidic
code necessary for Sec24-CFTR association and ER exit and thus is
retained in the ER but is not misfolded (Wang et al., 2004;
Farinha et al., 2013). Finally, we tested RDR01752 in combination
with low temperature and the FDA-approved corrector drugs
VX-809/lumacaftor and VX-661/tezacaftor (and compound C18) with and
without chronic exposure of the potentiator VX-770/ivacaftor.