Probing Conformational Rescue Induced by a Chemical Corrector of F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutant

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause loss of function of the CFTR channel on the apical surface of epithelial cells. The major CF-causing mutation, F508del-CFTR, is misfolded, retained in the endoplasmic reticulum, and degraded. Small molecule corrector compounds have been identified using high throughput screens, which partially rescue the trafficking defect of F508del-CFTR, allowing a fraction of the mutant protein to escape endoplasmic reticulum retention and traffic to the plasma membrane, where it exhibits partial function as a cAMP-regulated chloride channel. A subset of such corrector compounds binds directly to the mutant protein, prompting the hypothesis that they rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis directly by evaluating the consequences of a corrector compound on the conformation of each nucleotide binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. Interestingly, we found that VRT-325 was capable of partially restoring compactness in NBD1. However, VRT-325 had no detectable effect on the conformation of the second half of the molecule. In comparison, ablation of the di-arginine sequence, R 553 XR 555 (F508del-KXK-CFTR), modified protease susceptibility of NBD1, NBD2, and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together, these interventions restored processing of F508del-CFTR to near wild type. Importantly, however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. Importantly, this defect in channel activation can be fully corrected by the addition of the potentiator, VX-770.

Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules

“…The VRT-325 compound appears to work by directly binding to the ΔF508 molecule (37) and by promoting interactions between the two halves of the ΔF508 CFTR molecule (52). Specifically, Yu et al (53) recently showed that VRT-325 stabilizes the ΔF508 CFTR NBD1 domain, protecting the isolated domain from limited proteolysis, but had no effect on the second half of the ΔF508 CFTR molecule. Thus, the VRT-325 molecule corrects in ΔF508 CFTR the exact defect that we describe here for Q141K ABCG2, and indeed, we found that VRT-325 significantly increased total, dimer, and surface expression of the Q141K protein.…”

Structurally Distinct Ligands Rescue Biogenesis Defects of the KATP Channel Complex via a Converging Mechanism

“…Many small molecules bind directly and specifically to the affected proteins, thus acting as pharmacological chaperones. Pharmacological chaperones are generally thought to exert their effects via ligand-assisted protein folding and stabilization (16,17,19). Although this concept is easy to understand in monomeric proteins or oligomeric proteins with similar subunits, it can have complex manifestations in heteromultimeric proteins like the K ATP channel, where not only folding of the mutant protein itself but also interactions with assembly partners need to be considered.…”

Repairing mutated proteins – development of small molecules targeting defects in the cystic fibrosis transmembrane conductance regulator

“…For its promising in vitro pharmacological characteristics, VRT-325 (8b) has been intensely investigated in a variety of studies. Several distinct experiments [34][35][36][37] suggest that the correcting activity of 8b and other CFTR correctors might at least in part be due to direct interaction of the compounds with the channel protein. VRT-325 (8b) reduced the protease susceptibility of the F508del-CFTR NBD1 in vitro, indicating that the compound might induce conformational correction of the defective NBD1 [37].…”