Profiling the CFTR Variant Selectivity and Off-Target Interactions of VX-121

More than 1,200 variants of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are associated with cystic fibrosis (CF), an autosomal recessive pulmonary disease affecting over 100,000 people. Most people with CF bear a common CFTR variant (F508del) that can be treated with therapeutics containing "correctors" that suppress the misfolding of the CFTR chloride channel. However, the pharmacological responsiveness of other rare CF variants can vary tremendously. The approval of VX-121, a VX-445 analog that serves as a key component of Alyftrek^™, potentially provides a new therapeutic option for those with rare CF variants. Nevertheless, it remains unclear whether VX-121 offers superior rescue across the entire spectrum of rare CF variants. In this work, we use deep mutational scanning (DMS) to survey the impact of VX-121 on the plasma membrane expression of 232 rare CF variants. Our results show that VX-121 generally enhances CF variant expression more than VX-445 and is most potent towards variants with mutations in the first membrane spanning domain (MSD1). However, we identify one variant (Y1032C) with diminished proteostatic and functional selectivity for VX-121 relative to VX-445. Computational docking suggests that the native Y1032 side chain forms favorable interactions with VX-121 that are disrupted by this mutation in a manner that alters its coordination. Finally, using photo-crosslinking, we show that VX-121 avoids a key off-target interaction of VX-445. Together, our findings provide new insights into the similarities and differences between current approved CF therapeutics.