Elsevier

Biochemical Pharmacology

Volume 86, Issue 5, 1 September 2013, Pages 612-619
Biochemical Pharmacology

Corrector VX-809 stabilizes the first transmembrane domain of CFTR

https://doi.org/10.1016/j.bcp.2013.06.028Get rights and content

Abstract

Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis (CF). A potential CF therapy would be to repair CFTR processing mutants. It has been demonstrated that processing mutants of P-glycoprotein (P-gp), CFTR's sister protein, can be efficiently repaired by a drug-rescue mechanism. Many arginine suppressors that mimic drug-rescue have been identified in the P-gp transmembrane (TM) domains (TMDs) that rescue by forming hydrogen bonds with residues in adjacent helices to promote packing of the TM segments. To test if CFTR mutants could be repaired by a drug-rescue mechanism, we used truncation mutants to test if corrector VX-809 interacted with the TMDs. VX-809 was selected for study because it is specific for CFTR, it is the most effective corrector identified to date, but it has limited clinical benefit. Identification of the VX-809 target domain will help to develop correctors with improved clinical benefits. It was found that VX-809 rescued truncation mutants lacking the NBD2 and R domains. When the remaining domains (TMD1, NBD1, TMD2) were expressed as separate polypeptides, VX-809 only increased the stability of TMD1. We then performed arginine mutagenesis on TM6 in TMD1. Although the results showed that TM6 had distinct lipid and aqueous faces, CFTR was different from P-gp as no arginine promoted maturation of CFTR processing mutants. The results suggest that TMD1 contains a VX-809 binding site, but its mechanism differed from P-gp drug-rescue. We also report that V510D acts as a universal suppressor to rescue CFTR processing mutants.

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7) is a cAMP-regulated chloride channel that is located on the apical surface of epithelial cells that line lung airways and ducts of various glands (reviewed in Ref. [1]). CFTR regulates salt secretion and reabsorption to maintain normal salt and water homeostasis in epithelial tissues [2].

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CFTR gene that impair synthesis and trafficking of the protein or cause reduced chloride channel activity [3]. The most common defect is deletion of Phe508 (ΔF508 CFTR) in the first nucleotide-binding domain (NBD1). ΔF508 is a processing mutation that inhibits folding in the endoplasmic reticulum (ER) and trafficking to the cell surface [4]. The lack of chloride channel activity in CF patients due to defects in CFTR leads to mucosal obstruction of a variety of ducts within organs such as the pancreas, liver, salivary glands, sweat glands and lungs [5]. The main cause of morbidity is the presence of thick tenacious secretions that obstruct distal airways and submucosal glands in the lung. CF patients have recurrent bouts of lung infections that result in a decline in respiratory function and eventual lung failure.

We discovered that a drug-rescue approach could be used to repair processing mutants of the P-glycoprotein (P-gp) drug pump, CFTR's sister protein [6]. Drug-rescue appeared to be a direct effect because it could be mimicked by introducing arginine suppressor mutations into the TMDs [7]. It was observed that the majority of arginines introduced into the faces of TM segments 6 or 12 predicted to line the aqueous channel promoted maturation of P-gp processing mutants. In addition, the TMDs of other ABC proteins appear to be targets to repair defects caused by mutations equivalent to ΔF508. For example, suppressor mutations in TM segments 2 and 12 rescued ΔY670 Yor1p (equivalent to ΔF508 CFTR), a yeast ABC drug transporter [8].

We hypothesize that it may also be possible to specifically rescue ΔF508 CFTR and other CFTR processing mutants by a direct drug-rescue approach using small molecules. Evidence that CFTR processing mutants could be repaired by a direct rescue approach is the observation that many second-site suppressors can rescue ΔF508-CFTR [9], [10]. Potential advantages of a direct rescue approach are that expression of proteins involved in other metabolic pathways would not be altered and rescue would be less affected by differences in cellular folding environments in different tissues or in different disease states. Intensive efforts have been made to screen chemical libraries for compounds (called correctors) that could rescue ΔF508-CFTR. Unfortunately, rescue by correctors identified to date appears to be too low for effective therapy [11], [12].

The most promising corrector identified to date is VX-809 [13]. VX-809 appeared to be specific, as it did not promote maturation of P-gp or hERG K+ channel processing mutants. Most other correctors promoted maturation of both CFTR and P-gp processing mutants [14]. The specificity of VX-809 suggests that it may bind directly to CFTR. In a clinical trial however, it was found that VX-809 only caused a small increase in sweat conductance and no increase in the levels of mature ΔF508-CFTR were observed in rectal biopsies [15]. Identification of the VX-809 target region in CFTR would aid in the development of more effective correctors. In this study, we tested whether VX-809 rescued CFTR processing mutants by a P-gp drug-rescue mechanism. We tested whether VX-809 affected the TMDs of CFTR and whether arginines introduced into the aqueous face of TM6 (the P-gp hotspot for arginine suppressors) would promote maturation of CFTR processing mutants.

Section snippets

Chemicals

Corrector 3-(6-{[1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonyl]-amino}-3-methyl-pyridin-2-yl)-benzoic acid (VX-809) was obtained from Selleck Chemicals LLC (Houston, TX). Dulbecco's modified Eagle's media and calf serum were obtained from Wisent Inc. (St. Bruno, Quebec). Monoclonal antibody against GAPDH was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody A52 and rabbit polyclonal antibody against CFTR were generated as described previously [16], [17].

Effect of VX-809 on maturation of CFTR truncation mutants

The 1480 amino acids of CFTR are organized into two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and an R domain [3] (Fig. 1A). Each homologous half contains an N-terminal TMD followed by an NBD. A secondary structure model is shown in Fig. 1B. The secondary structure predicts that each TMD is linked to each NBD via intracellular loops (ICLs). Domain interactions are predicted to be an important feature of CFTR maturation, inhibition of maturation by processing

Discussion

VX-809 is an important corrector as it is specific for CFTR and it is the most efficient corrector in promoting maturation of ΔF508 CFTR [13]. Most CFTR correctors are not specific as they will rescue processing mutants of different proteins. For example, most CFTR correctors will also rescue processing mutants of the P-gp drug pump [14]. Specificity is important because specific rescue of CFTR processing mutants would reduce side effects caused by alteration in expression levels of proteins

Acknowledgements

This study was supported by grants from Cystic Fibrosis Canada and the Canadian Institutes for Health Research (grant 62832). D.M.C. is the recipient of the Canadian Chair in Membrane Biology.

References (45)

  • T.W. Loo et al.

    Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)

    J Biol Chem

    (2008)
  • B. Kleizen et al.

    Folding of CFTR is predominantly cotranslational

    Mol Cell

    (2005)
  • T.W. Loo et al.

    Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions

    J Biol Chem

    (1998)
  • A.A. Aleksandrov et al.

    Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR

    J Mol Biol

    (2010)
  • T.W. Loo et al.

    Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11

    J Biol Chem

    (2008)
  • F.S. Seibert et al.

    Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity

    J Biol Chem

    (1996)
  • H.M. Sampson et al.

    Identification of a NBD1-binding pharmacological chaperone that corrects the trafficking defect of F508del-CFTR

    Chem Biol

    (2011)
  • Y. El Hiani et al.

    Changes in accessibility of cytoplasmic substances to the pore associated with activation of the cystic fibrosis transmembrane conductance regulator chloride channel

    J Biol Chem

    (2010)
  • T.W. Loo et al.

    Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding

    J Biol Chem

    (2003)
  • J.F. Cotten et al.

    Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge

    J Biol Chem

    (1999)
  • F.S. Seibert et al.

    Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity

    J Biol Chem

    (1996)
  • J.R. Riordan

    CFTR function and prospects for therapy

    Annu Rev Biochem

    (2008)
  • Cited by (0)

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