Influence of cell background on pharmacological rescue of mutant CFTR

Abstract

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue.

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

“…To date, C4 is one of the most well-studied correctors for F508del-CFTR rescue. However, the efficiency of rescue for C4 is quite limited across multiple cell types that have been tested (15,18,32). This result is in agreement with data obtained from both F508del-CFTR FAP constructs in HEK293 cells as well as the currents associated with F508del-CFTR endogenously expressed from HBE cells, where functional C4 correction efficacy was poor (Table 1).…”

“…The pattern of corrector efficacy was strikingly similar across the disparate cell types and assays that we examined, and this has not always been observed (15,16). Nevertheless, our results highlight the fidelity of the FAP reporter system to recapitulate the behavior of F508del-CFTR in a system that expresses the mutant untagged protein endogenously.…”

“…Accumulation of the C band after corrector treatment is quite low with untagged F508del-CFTR and can depend on the cell type examined (13,15). To date, C4 is one of the most well-studied correctors for F508del-CFTR rescue.…”

“…Thus far, correctors have shown limited ability to improve F508del-CFTR trafficking. Even after corrector treatment, F508del-CFTR has <15% the activity of CFTR wild-type (WT), and the corrector efficacy may vary depending on the cell type examined (13)(14)(15)(16). More robust rescue can be achieved by using a combination of correctors, which enhances rescue to levels greater than individual compound actions (17)(18)(19).…”

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809

“…S5). The ability of some CFTR correctors to act in recombinant cells expressing CFTR but not in native HBE cultures has been previously reported (33) and highlights the importance of confirming activity in native epithelial cells.…”