Functional and biochemical characterization of Alyftrek® components reveals high-potency rescue of F508del-CFTR.

Alyftrek® (vanzacaftor/tezacaftor/deutivacaftor, VTD) is a triple CFTR modulator therapy for patients with cystic fibrosis (CF). In this study, we characterized the pharmacological properties of vanzacaftor and deutivacaftor using functional and biochemical assays in patients derived primary bronchial epithelial (HBE) cells (F508del/F508del, F508del/R1162X, F508del/W1282X) and in CFBE41o⁻ cells expressing F508del-CFTR. Cells were treated with Alyftrek® components across a range of concentrations. The S- and R-enantiomers of vanzacaftor were evaluated separately, and the activity of deutivacaftor was compared with that of ivacaftor. CFTR function was assessed by short-circuit current measurements and whole-cell patch-clamp recordings, while CFTR protein maturation was analyzed by Western blotting. Treatment with S-vanzacaftor alone restored F508del-CFTR-dependent chloride currents with an EC₅₀ of 130 nM, which decreased markedly to 8 nM when combined with tezacaftor. Substitution of ivacaftor with deutivacaftor in triple combinations (VTI vs. VTD) yielded comparable functional responses. Both VTD and VTI promoted the concentration-dependent appearance of the mature, fully glycosylated CFTR C-band with an EC₅₀ of 11 nM. The R-enantiomer of vanzacaftor was markedly less potent in rescuing F508del-CFTR and antagonized the effect of the S-enantiomer. Concentration-reduction experiments further showed that 10% to 50% of the initial VTI/VTD concentrations were sufficient to achieve substantial F508del-CFTR rescue. Collectively, these findings demonstrate the high potency of S-vanzacaftor at nanomolar concentrations, both alone and in combination with tezacaftor. The potentiator deutivacaftor exhibited efficacy comparable to that of ivacaftor.