siRNA Screen Identifies Potential Cystic Fibrosis Drug Target

A large-scale screen combining high-content live-cell microscopy and small interfering RNAs (siRNAs) in human airway epithelial cells from patients with cystic fibrosis (CF) has pinpointed a promising target for drug treatment.

Cystic fibrosis is an inherited disease caused by a mutation in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that affects the body's ability to move salt and water in and out of cells. CTFR codes for an ABC transporter-class ion channel, ENaC, which transports chloride and thiocyanate ions across epithelial cell membranes. Mutations of the CFTR gene disrupt the functioning of ENaC chloride ion channels in these cell membranes, leading to cystic fibrosis.


All disease-causing mutations in the CFTR gene prevent the channel from functioning properly, leading to a blockage of the movement of salt and water into and out of cells. As a result of this blockage, cells that line the passageways of the lungs, pancreas, and other organs produce abnormally thick, sticky mucus. This mucus obstructs the airways and glands, causing the characteristic signs and symptoms of cystic fibrosis. While thin mucus can be removed by cilia, thick mucus cannot be removed by cilia, so it traps bacteria that give rise to chronic infections. The only drug currently available that directly counteracts a cystic fibrosis-related mutation only works on the 3% of patients that carry one specific mutation out of the almost 2000 CFTR mutations detected so far.

Investigators at the European Molecular Biology Laboratory (Heidelberg, Germany) and their colleagues at the University of Lisbon (Portugal) sought to develop a global understanding of molecular regulators of ENaC traffic and function and to identify candidate CF drug targets. To this end, they performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells.

A study published in the September 12, 2013, online edition of the journal Cell revealed that the investigators screened over 6,000 genes and identified over 1,500 candidates, evenly divided between channel inhibitors and activators.

Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of DGKi (diacylglycerol kinase, iota), a protein involved in phosphatidylinositol biphosphate (PiP2) metabolism, downgraded ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

“Inhibiting DGKi seems to reverse the effects of cystic fibrosis, but not block ENaC completely,” said senior author Dr. Margarida Amaral, professor of chemistry and biochemistry at the University of Lisbon, “indeed, inhibiting DGKi reduces ENaC activity enough for cells to go back to normal, but not so much that they cause other problems, like pulmonary edema.”

Related Links:
European Molecular Biology Laboratory
University of Lisbon