(648) Distinct Proteostasis and Structural States Drive VX-809 Susceptibility of CFTR Mutants

Background: C

Cystic Fibrosis (CF) is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel. Currently the only targeted CF treatment uses pharmacologic chaperones to thermodynamically stabilize mutant CFTR. The pharmacologic chaperone VX-809 transformed CF therapy by correcting F508del, the most common patient mutation. However, F508del exhibits only mild VX-809 response. By contrast, rarer mutations P67L and L206W demonstrate a hyper-response to VX-809¹. Here, we address the mechanisms underlying different susceptibility to VX-809 for different CFTR mutations.

Methods: We used quantitative affinity purification mass spectrometry-based interactomics to characterize VX-809 hyper-responsive P67L and L206W and mildly responsive F508del². We co-immunoprecipitated CFTR and measured proteostasis interactions using Tandem Mass Tag labeling based quantitative mass spectrometry. This method allows for direct comparison of interactor levels between mutants and drug treatment condition. To understand the structural contributions, we developed a computational approach for simulating CFTR conformational space in silico³. We use cryo-EM density data refinement and Rosetta comparative modeling (RosettaCM) and benchmarked our simulations against experimental thermodynamic data. We then modeled multi-domain WT, ΔF508, P67L, and L206W CFTR using RosettaCM with and without VX-809.

Results: Using quantitative interactomics, we determined VX-809 creates an inflection point in proteostasis interactions between folding and degradation machinery for P67L and L206W compared to F508del. Degradation occurs through the proteasome and autophagy pathways; thus, we inhibited the proteasome and lysosome respectively and measure the change in the VX-809 response of each mutant. Inhibiting the proteasome attenuated P67L and L206W VX-809 response, suggesting a previously unidentified role for protein degradation in VX-809 correction. Our structural models reveal distinct structural defects experienced by hyper-responsive mutants P67L and L206W and offer clinically relevant information about CFTR VX-809 mechanisms.

Conclusions: We conclude that different CFTR mutants experience distinct proteostasis states that drive VX-809 susceptibility, specifically interactions with protein folding and degradation machinery. Furthermore, we hypothesize specific structural defect may predispose mutants to corrector susceptibility and in turn influence CFTR proteostasis.

Acknowledgements: The Meiler Lab was funded by NIH #1R01GM080403, the Plate Lab was funded by NIH #1R35GM133552, EFM was funded by training grant NIH #5T32GM065086.

References: [1] Sabusap CM, Wang W, McNicholas CM, Chung WJ, Fu L, Wen H, et al. Analysis of cystic fibrosis–associated P67L CFTR illustrates barriers to personalized therapeutics for orphan diseases. JCI Insight 2016;1:1–10. https://doi.org/10.1172/jci.insight.86581.

[2] McDonald EF, Sabusap CMP, Kim M, Plate L. Distinct proteostasis states drive pharmacologic chaperone susceptibility for Cystic Fibrosis Transmembrane Conductance Regulator misfolding mutants. BioRxiv 2021:2021.09.09.459524. https://doi.org/10.1101/2021.09.09.459524.

[3] McDonald EF, Woods H, Smith ST, Kim M, Schoeder CT, Plate L, et al. Structural Comparative Modeling of Multi-Domain F508del CFTR. Biomol 2022. https://doi.org/10.3390/biom12030471.