Kalydeco’s Efficacy Tied to Phosphorylation Levels of CFTR Protein, Study Suggests

CFTR channel activity is controlled by a molecular process known as phosphorylation. Now, a study suggests that the levels of phosphorylation affect how well Kalydeco works in people with certain mutations in the CFTR gene.

The study “VX-770-mediated potentiation of numerous human CFTR disease mutants is influenced by phosphorylation level” was published in the journal Nature Scientific Reports.

More than 2,000 mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, with evidence linking 300 of these mutations with the development of cystic fibrosis (CF).

The gene has instructions for the production of the CFTR protein, which serves as a gate at the cell surface, opening to allow chloride ions to cross the cell membrane. The opening and closing of the CFTR channel is regulated by the addition of a chemical group, called phosphate, in the regulatory domain (R domain) of the channel. This process, called phosphorylation, is mediated by two proteins called PKA and PKC.

Kalydeco (ivacaftor), marketed by Vertex Pharmaceuticals and previously known as VX-770, is an approved CF therapy for patients ages 12 months and older, carrying up to 38 specific mutations in the CFTR gene, including rare mutations (also known as ultra-orphan mutations).

The therapy works by keeping the CFTR gate open longer at the cell surface, easing the transport of salts and water in and out of cells to improve hydration and mucus clearance. In other words, it works as a potentiator to enhance the activity of the CFTR protein.

Researchers at Emory University School of Medicine investigated whether Kalydeco’s therapeutic performance depends on the phosphorylation of the CFTR protein.

In an in vitro experiment, they injected female frog eggs (called oocytes) with genetic material, namely messenger RNA — the template that cells use to make proteins — encoding the information to produce a human normal (wild-type) or mutated CFTR protein. The oocytes were then treated with a low (6.4 U/ml) or a high (127.6 U/ml) concentration of the PKA protein.

Results showed that PKA-mediated phosphorylation of wild-type CFTR affected how Kalydeco potentiates CFTR function. Wild-type CFTR function increased in a dose-dependent manner with PKA, “possibly by increase channel open probability and the number of active channels,” the researchers wrote.

In turn, in three disease-associated CFTR mutants — mutations E193K-, K1060T-, and N1303K — located in different regions of the CFTR gene, researchers saw that Kalydeco increased the activity of mutated CFTR by 30% when in the presence of high phosphorylation levels (with 127.6 U/ml PKA). But the effect was stronger when phosphorylation levels were lower (6.4 U/ml PKA), with Kalydeco potentiating the mutated CFTR by 100%.

“These data provide strong evidence to support our hypothesis that VX-770 [Kalydeco] potentiates wildtype as well as disease-associated mutants in a graded phosphorylation-dependent manner,” the researchers wrote.

The potentiation of other CFTR mutants, including the G551D mutation, was also observed, but was less dependent upon the level of phosphorylation.

In the case of the F508del mutation, the most common mutation in CF patients, Kalydeco action was also less dependent on phosphorylation.

Testing their findings on human primary bronchial epithelial cells, the researchers observed that Kalydeco’s action was indeed dependent on phosphorylation status of the CTFR protein.

Overall, the results show that phosphorylation affects the activity of CFTR and, consequently, the action of potentiators like Kalydeco. According to the team, this has clear implications when screening for potential therapeutics.

“The efficacy of potentiators may be obscured by a ceiling effect when drug screening is performed under strongly phosphorylating conditions,” the researchers wrote. “These results should be considered in campaigns for CFTR potentiator discovery.”

Further studies are required to “fully understand the mechanism and binding sites of VX-770 in potentiation of CFTR to benefit the CF patient community,” the team concluded.