(647) Potentiator Icenticaftor Is Superior to Ivacaftor in Rescuing F508del CFTR and Does Not Destabilize Corrected Mature Protein

Background: Cystic fibrosis (CF) is caused by mutations in the ion channel, CFTR, which is required for proper hydration of airway epithelial surfaces that prevents hyperconcentration of mucus, infection, and inflammation. Over 2000 mutations in CFTR have been identified. Rescue of the most common mutation in CF, F508del, and other mutant CFTR proteins is now feasible with the triple therapy, Trikafta, consisting of two corrector compounds, elexacaftor (elx) and tezacaftor (tez), plus potentiator ivacaftor (iva). While iva activates corrected F508del, we and others have observed that chronic treatment reduced the levels of mature F508del by enhancing its turnover [1,2,3]. Here we test the novel potentiator compound icenticaftor/QBW251 (icn) that is currently in clinical trials for COPD [4,5,6], for its ability to enhance rescued CFTR function.

Methods: Non-CF, F508del/F508del, and F508del/G542X primary human bronchial epithelial (HBE) cultures and the cell line, BHK-21, expressing wild-type or F508del CFTR were treated with correctors and potentiators to compare CFTR maturation and function. HBE were mounted in Ussing chambers to determine ion channel function of rescued CFTR, and cell lysates were analyzed by Western blot to detect CFTR maturation levels. BHK-21 cells expressing wild-type or F508del CFTR were used for single-channel measurements in the lipid bilayer.

Results: Acute treatment of icn in Ussing chambers enhances function of wild-type CFTR in non-CF HBE in a dose-dependent fashion, starting at concentrations as low as 0.1 uM, before and after addition of forskolin. In the presence of elx, CF HBE (F508del/G542X) rescue of F508del is enhanced upon acute addition of icn, and most effective when added before forskolin. Notably, in F508del/F508del CF HBE cells, a 24 h treatment using a modified triple, elx/tez/icn, led to a superior functional rescue when compared to the Trikafta triple. Wild-type CFTR open probability (Po) in single-channel measurements after addition of icn was similarly improved as after addition of iva. Consistent with results in Ussing chambers, single-channel measurements indicated a superior restoration of function of F508del by elx/tez/icn compared with elx/tez/iva (Po at 37⁰C improved twofold over iva). Furthermore, the destabilization of mature F508del that was observed with iva, was not observed with icn and the mature corrected F508del protein levels were three times higher with icn.

Conclusions: Based on our results, we can conclude that icn is a superior CFTR modulator when compared to iva as it enhances function but does not destabilize rescued F508del, thus leading to improved function. These data are critical for considering development of advanced therapies. Understanding the different mechanisms of CFTR potentiator activities may provide further benefits for CF patients with F508del and other mutations. Future studies will address whether elx/tez/icn rescues rare mutations with enhanced efficacy to optimize therapeutics for all CF patients.

Acknowledgements: Supported by CFF (BOUCHE19R0) and NIH (P30DK065988).

References: 1) Cholon et al. Sci Transl Med. 2014 6(246):246ra96

2) Veit et al. Sci Transl Med. 2014 Jul 23;6(246):246ra97

3) Becq et al. Eur Respir J. 2022 59(2):2100671

4) Grand et al., J Med Chem. 2021 64(11):7241-7260

5) Kazani et al. J Cyst Fibros. 2021 20(2):250-256.

6) Rowe et al. Int J Chron Obstruct Pulmon Dis. 2020;15:2399-2409