New horizons in the treatment of cystic fibrosis

Abstract

Cystic fibrosis (CF) is a lethal, recessive, genetic disease affecting approximately 1 in 2500 live births among Caucasians. The CF gene codes for a cAMP/PKA-dependent, ATP-requiring, membrane chloride ion channel, generally found in the apical membranes of many secreting epithelia and known as CFTR (cystic fibrosis transmembrane conductance regulator). There are currently over 1700 known mutations affecting CFTR, many of which give rise to a disease phenotype. Around 75% of CF alleles contain the ΔF508 mutation in which a triplet codon has been lost, leading to a missing phenylalanine at position 508 in the protein. This altered protein fails to be trafficked to the correct location in the cell and is generally destroyed by the proteasome. The small amount that does reach the correct location functions poorly. Clearly the cohort of patients with at least one ΔF508 allele are a major target for therapeutic intervention. It is now over two decades since the CF gene was discovered and during this time the properties of CFTR have been intensely investigated. At long last there appears to be progress with the pharmaco-therapeutic approach. Ongoing clinical trials have produced fascinating results in which clinical benefit appears to have been achieved. To arrive at this point ingenious ways have been devised to screen very large chemical libraries for one of two properties: (i) agents promoting trafficking of mutant CFTR to, and insertion into the membrane, and known as correctors or (ii) agents which activate appropriately located mutant CFTR, known as potentiators. The best compounds emerging from these programmes are then used as chemical scaffolds to synthesize other compounds with appropriate pharmaceutical properties, hopefully with their pharmacological activity maintained or even enhanced. In summary, this approach attempts to make the mutant CFTR function in place of the real CFTR. A major function of CFTR in healthy airways is to maintain an adequate airway surface liquid (ASL) layer. In CF the position is further confounded since epithelial sodium channels (ENaC) are no longer regulated and transport salt and water out of the airways to exacerbate the lack of ASL. Thus an additional possibility for treatment of CF is to use agents that inhibit ENaC either alone or as adjuncts to CFTR correctors and/or potentiators. Yet a further way in which a pharmacological approach to CF can be considered is to recruit alternative chloride channels, such as calcium-activated chloride channel (CaCC), to act as surrogates for CFTR. A number of P2Y(2) receptor agonists have been investigated that operate by increasing Ca(2+)(i) which in turn activates CaCC. Some of these compounds are currently in clinical trials. The knowledge base surrounding the structure and function of CFTR that has accumulated in the last 20 years is impressive. Translational research feeding from this is now yielding compounds that provide real prospects for a pharmacotherapy for this disease.

Figure 1

This Figure summarizes the main differences in the transport of salts (and in consequence osmotic fluid movement) in healthy and cystic fibrosis (CF) airways. The diagram represents the processes at both the surface-ciliated epithelium and in submucosal glands (containing the secretory serous acini and mucus tubules). In normal airways, chloride and bicarbonate ions are secreted into the airway surface liquid (ASL, shown in blue) by an electrogenic process, while at the same time sodium ions are absorbed from the ASL, also by an electrogenic mechanism. Of course, counterions will move passively as a result of these processes. Whether fluid (water) moves from the ASL into the submucosal space or in the opposite direction will depend on the overall osmotic balance established by the two transport processes. The acini of the submucosal glands also secrete anions, probably by a process similar to that of the surface epithelium. The secretion also contains a variety of antimicrobial peptides, concerned with defence mechanisms and mucins (shown in red) are added as the fluid moves onto the airway surface. The balance of these processes is such that they maintain an ASL with a well-defined sol layer in contact with the epithelial surface and a viscous gel layer above, an ideal way in which to trap particulate matter in mucus and move it, by ciliary action, towards the exterior. In CF neither the surface epithelium nor the submucosal glands are capable of generating anion secretion because of the lack of functioning cystic fibrosis transmembrane conductance regulator, while sodium absorption is enhanced. The osmotic balance is altered such that fluid leaves the airway surface and moves into the submucosal space. In consequence, submucosal gland ducts are blocked by mucus, which also settles down on the cilia making them unable to function and bacterial infection establishes itself, aggravated by the loss of the antimicrobial peptides.

Figure 2

The different components of solute and fluid movement in airway tissue. All the data are derived from experiments on ovine tracheal tissue. (A) shows a recording of short circuit current (SCC), a measure of the total electrogenic transport of cations and anions across the epithelium. As cations are absorbed from the apical surface while anions are secreted in the opposite direction, these two currents will be additive. Sodium absorption can be completely eliminated by adding amiloride (Amil) to the apical surface and shows under the conditions of the experiment that it accounts for ∼75% of the total current. The remaining current, due largely to anion secretion, can be further enhanced, in this instance by the EP₄ receptor agonist L-902688. Data in (B) shows that pattern seen in (A) is confirmed. These results suggest that in the basal state the airway surface epithelia are absorptive. However, it is to be remembered that the in vivo condition is very different. First, under SCC conditions, the apical surface is bathed in a solution of infinite volume, compared to the airway surface liquid (ASL), and second, the airway epithelium is removed from the humoral and neural mechanisms present in vivo. However, there is a second source of fluid that contributes to the ASL and derived from the submucosal glands. To measure secretion from individual glands the method first described by Joo et al. 2001 was used. A small area of tracheal mucosa was mounted in a special chamber and bathed on the basolateral side with Krebs solution at 37°C and superfused with 95%O₂/5%CO₂. The apical surface, after cleaning and drying, was covered with a thin layer of water saturated mineral oil. Secretion from individual glands collected as spherical droplets at the mouth of the glands. A sequence of photographs of the airway surface at timed intervals allowed the secretion rate to be calculated using ImageJ software. (C) shows two views of the epithelial surface (10 mm², corner of grid with 0.5 mm squares is shown) of an ovine trachea before (upper) and a few minutes after (lower) carbachol had been added to the basolateral fluid. Note the appearance of spherical droplets at the surface opening of each gland.

Figure 3

The principles of high throughput screening for cystic fibrosis transmembrane conductance regulator (CFTR) potentiators. Epithelial cells expressing ΔF508 CFTR (shown in red) and also a halide sensitive yellow fluorescent protein (yellow cells), are grown in 100-well plates. As the screen is for potentiator molecules, the CFTR has first to be assisted to the membrane. By incubation at 27°C for 24 h transfer is achieved. Test compounds are added to the wells and here are shown to open channels. This is measured by changing chloride for iodide in the bathing solution. A rapid loss of fluorescence is indicative of a strong potentiator. Note in the absence of compounds there will be some loss of fluorescence. Also by including a standard compound, such as genistein, weaker compounds can be eliminated. The method described is capable of adaptation. For example, if the search is for CFTR correctors, then the 27°C stage would be eliminated. Also a variety of different cell lines can be used and in some instances a primer may be required, such as a low concentration of forskolin.

Figure 4

(A) shows the chemical structure of the cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770, currently in Phase 3 clinical trial. (B) illustrates the path followed to obtain the CFTR corrector compound VRT-325. Note the original library had 164 000 compounds of which 1028 were selected through high throughput screening, but only 18% survived retesting. One of the better compounds, VRT-442 had an EC₅₀ in the µM range and gave a correction equivalent to 47% of that achievable by temperature lowering to27°C. A further 500 analogues using this scaffold were then prepared and VRT-325 selected. Note that in VRT-325, the EC₅₀ is barely changed, yet it can achieve a correction of 74% of that of the temperature shift.