Biogenesis of γ-secretase early in the secretory pathway

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology

The Primary Folding Defect and Rescue of ΔF508 CFTR Emerge during Translation of the Mutant Domain

In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis

Correcting CFTR folding defects by small-molecule correctors to cure cystic fibrosis

Pharmacological intervention to treat the lethal genetic disease cystic fibrosis has become reality, even for the severe, most common folding mutant F508del CFTR. CFTR defects range from absence of the protein, misfolding that leads to degradation rather than cell-surface localization (such as F508del), to functional chloride-channel defects on the cell surface. Corrector and potentiator drugs improve cell-surface location and channel activity, respectively, and combination therapy of two correctors and a potentiator have shown synergy. Several combinations are in the drug-development pipeline and although the primary defect is not repaired, rescue levels are reaching those resembling a cure for CF. Combination therapy with correctors may also improve functional CFTR mutants and benefit patients on potentiator therapy

Correcting CFTR folding defects by small-molecule correctors to cure cystic fibrosis

Pharmacological intervention to treat the lethal genetic disease cystic fibrosis has become reality, even for the severe, most common folding mutant F508del CFTR. CFTR defects range from absence of the protein, misfolding that leads to degradation rather than cell-surface localization (such as F508del), to functional chloride-channel defects on the cell surface. Corrector and potentiator drugs improve cell-surface location and channel activity, respectively, and combination therapy of two correctors and a potentiator have shown synergy. Several combinations are in the drug-development pipeline and although the primary defect is not repaired, rescue levels are reaching those resembling a cure for CF. Combination therapy with correctors may also improve functional CFTR mutants and benefit patients on potentiator therapy

Mutations in the second cytoplasmic loop of CFTR suggest distinct mode of action between potentiators VX-770 and glpg1837

The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified a novel potentiator GLPG1837 by compound screening on mutant CFTR. YFP-halide efflux assays and single channel measurements showed ∼2.5-fold improvement in channel activity by GLPG1837 compared to VX-770 (ivacaftor/Kalydeco) on G551D CFTR (1, 2). GLPG1837 successfully passed the Phase-2 clinical trials and proved to be the first potentiator after VX-770 to show competitive results on G551D patients. To identify potential differences in the mode of actions of these potentiators we studied their effects on CFTR folding and function. Biochemical radiolabeling experiments showed that mutations in the intracellular loop 2 (ICL2) disrupt domain assembly between TMD1 and NBD2, a late folding event in CFTR, but in most cases do not impair CFTR trafficking towards the cell surface. Protease-susceptibility assays showed that VX-770 improved late TMD1 folding of many ICL2 mutations, but GLPG1837 did not. YFP-halide efflux assays showed that these ICL2 mutants had varying effect on channel function, ranging from wild-type-like to function-defective mutants. GLPG1837 restored function of non-CF gating mutant E267K much better than VX-770. Residue E267 in ICL2 electrostatically interacts with K1060 in ICL4 to promote channel opening (3). This indicates that GLPG1837 is more efficient in compensating for this lost interaction. Altogether, our biochemical and functional data suggests that potentiators VX-770 and GLPG1837 have a different mode of action

Deciphering the mode of action of clinically relevant next generation c2 corrector compounds GLPG2737 and GLPG3221

The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified C2 correctors by high-throughput compound screening and Med Chem optimization for cell surface rescue of F508del-CFTR. These C2 correctors are acting synergistically with a type I corrector such as ABBV/GLPG2222. Two C2 correctors, ABBV/GLPG2737 and ABBV/ GLPG3221 were optimized for drug like properties and are in clinical and pre-clinical evaluation, respectively. From both the functional halide efflux assays and pulse chase analysis we showed that the rescue efficiency of F508del-CFTR after combination treatment (C1 + C2) is markedly higher (≥50% of wild-type levels) than the sum of C1 and C2 correction. These strong synergistic effects show not only that C1 and C2 have a different mode of action, but also highlight the benefit of the triple-combination treatment with addition of a potentiator. To investigate how, when and where these C2 correctors act on CFTR we use radiolabeling approaches in combination with protease susceptibility assays. We first evaluated C1 corrector ABBV/GLPG2222 using in vitro translation and translocation assays in the presence of semi-intact HEK293 cells as source for endoplasmic reticulum (ER) membranes. We found that ABBV/GLPG2222, but not the C2 correctors, acted on transmembrane domain 1 (TMD1) in an identical fashion as lumacaftor by promoting its cytoplasmic loop packing important for domain folding. Varying the time of drug addition in pulse chase experiments showed that, like C1 corrector, both C2 correctors reached maximal rescue efficiency when present during, and shortly after the 15-minute pulse labelling. The C2 correctors acted additively with all F508del suppressors (I539T, G550E and R1070W) and did not restore nucleotide binding domain 1 (NBD1) folding in the F508del-CFTR background. Although we did not identify yet where the C2 correctors act, these compounds restored trafficking of the NBD2-less F508del-CFTR (F508del-1219X) construct very well. Our results show that the C2 correctors promote the earliest folding events of the ER-export competent CFTR molecule lacking NBD2, ruling out all possible NBD2 inter-domain assembly events (TMD1/NBD2; NBD1/NBD2; TMD2/NBD2) as target candidate. The triple-combination treatment that includes these C2 correctors significantly raises the F508del-CFTR rescue ceiling, with the aim to reach sufficient clinical benefit for most CF patients in the near future

CFTR: New insights into structure and function and implications for modulation by small molecules

Structural biology and functional studies are a powerful combination to elucidate fundamental knowledge about the cystic fibrosis transmembrane conductance regulator (CFTR). Here, we discuss the latest findings, including how clinically-approved drugs restore function to mutant CFTR, leading to better clinical outcomes for people with cystic fibrosis (CF). Despite the prospect of regulatory approval of a CFTR-targeting therapy for most CF mutations, strenuous efforts are still needed to fully comprehend CFTR structure-and-function for the development of better drugs to enable people with CF to live full and active lives