Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis

One of the major challenges facing the pharmaceutical field is the identification of novel, ‘druggable’ targets common to distinct diseases that, despite their clinical diversity, share the same basic molecular defect(s) – thus, being termed ‘horizontal diseases’. Membrane proteins constitute one of the largest families in the human genome and, given their major roles in cells and organisms, they are relevant to common human disorders such as cardiovascular disease and cancer, but also to rare genetic conditions such as cystic fibrosis (CF). Here, we review therapeutic approaches to correcting the basic defect in CF, which is caused mainly by the intracellular retention of a misfolded protein, and focus on various recent drug-discovery strategies for this important and paradigmatic disease. These strategies have possible applications in many membrane protein disorders, including other channelopathies. The mechanisms of action of potent and specific compounds, representing promising drug leads for CF pharmacotherapy, are explained and discussed.

Introduction

One of the major challenges facing the pharmaceutical field is the identification and validation of novel, high-quality, ‘druggable’ targets common to distinct diseases that, despite being clinically diverse, share the same basic molecular defect(s). These are termed ‘horizontal diseases’ because of their common defective cellular phenotype(s). Examples include misfolding and trafficking disorders.

Membrane proteins constitute some of the largest families encoded in the human genome, including ATP-binding cassette (ABC) transporters, ion channels and G-protein-coupled receptors (GPCRs). Given their major roles in cells and organisms, from transport and communication to immunity and nervous system functions, membrane proteins are highly relevant to frequent human disorders such as cardiovascular and neurological disorders, cancer, chronic pain, obesity and diabetes, but also to rare genetic conditions [1]. GPCRs and ion channels are currently the most attractive targets for drug discovery 2, 3.

Here, we provide a concise, up-to-date review of therapeutic approaches to cystic fibrosis (CF), with potential application in a wide range of diseases involving membrane proteins, particularly ABC transporters (see http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mono_001.chapter.137). These proteins are central to cancer (resistance to drugs), metabolic disorders (hyperinsulinemia, dyslipidemias and lysosomal disorders), osteoporosis, hearing loss and many other disorders [4].

CF is the most common lethal monogenic disorder in Caucasians, estimated to affect one per 2500–4000 newborns [5]. Clinically, CF is dominated by chronic lung disease, which is the main cause of morbidity and mortality. Airway obstruction by thick mucus and chronic infection by Pseudomonas aeruginosa eventually lead to loss of pulmonary function [6]. Other CF symptoms include pancreatic dysfunction, elevated sweat electrolytes and male infertility.

CF is caused by dysfunction of a single gene encoding the CF transmembrane conductance regulator (CFTR), which is an ABC transporter (ABCC7) that functions as a Cl⁻ channel in the apical membrane of epithelial cells lining the target organs (lung, intestine and sweat gland). CFTR, probably the best-studied ion channel, is regulated by cAMP-dependent protein kinase (PK)A and ATP. Its 1480 amino acids constitute two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs) and one regulatory domain (RD), which undergoes multiple phosphorylations [7]. Despite impressive advances in elucidating the molecular basis of CF, most current therapies are still restricted to alleviating symptoms [8]. Thus, life expectancy and quality of life, although largely improved recently, are still limited for CF patients [6].

Approaches aimed at correcting the basic CF defect still hold promise for curing the disease. But what is the basic defect in CF? To date, ∼1500 CFTR mutations have been described, most of which cause CF (see the CFTR Mutation Database: http://www.genet.sickkids.on.ca/cftr/app). Between these gene defects and the ultimate clinical phenotype of respiratory insufficiency, several events comprise the so-called ‘CF pathogenesis cascade’ (Figure 1a).

To correct such a variety of gene and protein defects effectively, CFTR mutants are grouped into functional classes (Figure 2b) that can be corrected through the same restoring strategy – an approach termed ‘mutation-specific therapy’ [9] (Box 1).

The emphasis of these strategies is on a single mutation, F508del (included in class II; Figure 1b and Box 1), because it occurs in ∼90% of CF patients. Accordingly, many efforts have focused on elucidating the molecular mechanism of F508del-CFTR dysfunction. CFTR without residue F508 is a particularly difficult folding substrate, the abnormal conformation of this mutant being recognized and retained by the endoplasmic reticulum quality control (ERQC), which rapidly targets it for proteasomal degradation 10, 11. Thus, very little or no F508del-CFTR, depending on the cell type, reaches the cellular membrane. Nevertheless, rescuing this mutant to the cell surface would be therapeutically relevant because it retains some Cl⁻ channel function. The ERQC acting on F508del-CFTR has also been a good model with which to describe the key players of the ERQC – mostly molecular chaperones such as Hsp70/Hdj-2, calnexin, CHIP, Hsp90 and Hdj-1/Hsp40 (for review, see Ref. [10]) – and the mechanisms of retention and degradation, which are likely to be shared by several other mutant proteins 12, 13.

Here, we focus on two types of recent and promising compound, emphasizing their mechanism of action: (i) compounds that rescue the trafficking defect of class II mutations (‘correctors’); and (ii) compounds that overcome or enhance the defective Cl⁻ channel activity of class III, IV and V CFTR mutants (‘potentiators’).

Because CFTR, in addition to being a channel, regulates epithelial ion transport by interfering with several other ion channels and transporters, we also describe a ‘bypassing’ approach to tackling drug discovery for CF. This strategy aims to correct the ionic imbalance in CF, which contributes to CF lung pathogenesis [14], through stimulation of bypassing ionic pathways that might compensate for the absence of functional CFTR.