Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein

*Department of Biochemistry and Biophysics.
The FASEB Journal (Impact Factor: 5.04). 10/2012; 27(2). DOI: 10.1096/fj.12-216119
Source: PubMed


Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T(1/2)∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

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Available from: Andrei A Aleksandrov, Jan 07, 2015
    • "Accordingly, the lower processing efficiency of C3 and C4 may result from the absence of this stabilization which does not occur for these two molecules. It was previously shown that VX-809 does not lower the turnover of F508del- CFTR to a rate similar to its wt counterpart (He et al. 2013). However, our data shown here, demonstrate that there is a significant decrease in comparison to nontreated cell lines. "
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    ABSTRACT: Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.
    08/2015; 3(4):e00152. DOI:10.1002/prp2.152
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    • "Please cite this article as: He Lihua, et al, Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ΔF508 CFTR Folding and Assembly, J Mol Biol (2014), Turning to the impact of modifying the NBD1/CL4 interface, which may be a site of action of the VX-809 corrector [23] [25], the R1070W mutation, shown by others to improve ΔF508 CFTR maturation [8], was the main tool we employed. We observed that the mutation alone promoted only a low level of maturation as did the other known interface change, V510D [43]. "
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    ABSTRACT: CFTR (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis (CF). The most common CF-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question we varied the extent of stabilization of NBD1 using different second site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ∆F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.
    Journal of Molecular Biology 07/2014; 427(1). DOI:10.1016/j.jmb.2014.07.026 · 4.33 Impact Factor
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    • "In the following experiments, we investigated the effect of temperature on the activity of F508del-CFTR. Thermal inactivation or instability of the mutant F508del at 37°C was recently shown in mammalian cells (Wang et al., 2011; He et al., 2013) and Xenopus oocytes (Liu et al., 2012). "
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    ABSTRACT: The mutated protein F508del-CFTR failed to traffic properly due to its retention in the ER and function as a Cl- channel with abnormal gating and endocytosis. Small chemicals (called correctors) individually restore F508del-CFTR trafficking and Cl- transport function but recent findings speak for taking into account synergistic pharmacology to better address CFTR defects. Here, we studied the function and maturation of F508del-CFTR expressed in HeLa cells with combination of five correctors (miglustat, IsoLAB, Corr4a, VX809 and SAHA). Using the whole-cell patch-clamp technique, the current density recorded in response to CFTR activators (forskolin+genistein) was significantly increased in presence of the following combinations: VX809+IsoLAB; VX809+miglustat+SAHA; VX809+miglustat+IsoLAB; VX809+IsoLAB+SAHA; VX809+miglustat+IsoLAB+SAHA. These combinations restored the activity of F508del-CFTR but with differential effect on the appearance of mature c-band of F508del-CFTR proteins. Focusing on the VX809+IsoLAB cocktail, we recorded a level of correction higher at 37°C versus room temperature, but without amelioration of the thermal instability of CFTR. The level of functional rescue with VX809+IsoLAB after 4h of incubation was maximal and similar to the one obtained in optimal conditions of use for each compound (i.e. 24h for VX809 + 4h for IsoLAB). Finally we compared the stimulation of F508del-CFTR by forskolin or forskolin+VX770 with cells corrected by VX809+IsoLAB. Our results open new perspectives for the development of a synergistic poly-pharmacology to rescue F508del-CFTR, show the importance of temperature on the effect of correctors and on the level of correction suggesting that optimized combination of correctors could lead to a better rescue of F508del-CFTR function.
    Journal of Pharmacology and Experimental Therapeutics 06/2014; 350(3). DOI:10.1124/jpet.114.214890 · 3.97 Impact Factor
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