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.