Ariel Roldan

Mechanism-based corrector combination restores ΔF508-CFTR folding and function

The most common cystic fibrosis mutation, ΔF508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and an incompletely understood mechanism, hampering drug development. Given the effect of second-site suppressor mutations, robust ΔF508-CFTR correction most likely requires stabilization of NBD1 energetics and the interface between membrane-spanning domains (MSDs) and NBD1, which are both established primary conformational defects. Here we elucidate the molecular targets of available correctors: class I stabilizes the NBD1-MSD1 and NBD1-MSD2 interfaces, and class II targets NBD2. Only chemical chaperones, surrogates of class III correctors, stabilize human ΔF508-NBD1. Although VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional plasma membrane expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in cystic fibrosis bronchial epithelial cells and intestinal organoids. Thus, the combination of structure-guided correctors represents an effective approach for cystic fibrosis therapy.

Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain

The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508‐CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508‐NBD1 and housekeeping proteins prevents ΔF508‐CFTR delivery to the plasma membrane. Based on this assumption we applied structure‐based virtual screening to identify new low‐molecular‐weight compounds that should bind to ΔF508‐NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico‐selected compounds induced functional expression of ΔF508‐CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508‐CFTR mice. The proposed compounds disrupt keratin8‐ΔF508‐CFTR interaction in ΔF508‐CFTR HeLa cells. Structural analysis of ΔF508‐NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508‐CFTR trafficking defect known to date.

Synergy-Based Small-Molecule Screen Using a Human Lung Epithelial Cell Line Yields ΔF508-CFTR Correctors That Augment VX-809 Maximal Efficacy

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium–derived cell line (CFBE41o−) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the “therapeutic ceiling” of first-generation correctors.

A HIV-1 Minimal Gag Protein Is Superior to Nucleocapsid at in Vitro Annealing and Exhibits Multimerization-induced Inhibition of Reverse Transcription

HIV-1 uses \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} to prime reverse transcription of its viral RNA. In this process, the 3′-end of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} must be annealed to the primer binding site of HIV-1 genomic RNA, and the two molecules together form a complex structure. During annealing, the nucleocapsid (NC) protein enhances the unwinding of tertiary structures within both RNA molecules. Moreover, the packaging of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} occurs prior to viral budding at a time when NC is still part of the Pr55Gag polyprotein. In contrast, Pr55Gag is able to produce virus-like particles on its own. We have recently shown that an N-terminal extended form of NC (mGag), containing all of the minimal elements required for virus-like particle formation, possesses greater affinity for HIV-1 genomic RNA than does NC alone. We have now studied the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}-annealing properties of mGag in comparison to those of NC and report that the former is more efficient in this regard than the latter. We have also tested each of a mutant version of mGag, an extended form of mGag, and an almost full-length form of Gag, and showed that all of these possessed greater tRNA-annealing capacity than did the viral NC protein. Yet, surprisingly, multimerization of Gag-related proteins did not abrogate this annealing process but rather resulted in dramatically reduced levels of reverse transcriptase processivity. These results suggest that the initial stages of reverse transcription may be regulated by the multimerization of Pr55Gag polyprotein at times prior to the cleavage of NC.

Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50–60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys447, obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system.

Chaperones rescue the energetic landscape of mutant CFTR at single molecule and in cell.

Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (∆F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 °C and concomitant functional inactivation of ∆F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at single-molecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart. Thus, molecular chaperones can contribute to functional maintenance of ∆F508-CFTR by reshaping the conformational energetics of its final fold, a mechanism with implication in the regulation of metastable ABC transporters and other plasma membrane proteins activity in health and diseases.The F508 deletion (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF causing mutation. Here the authors show that cytosolic chaperones shift the F508del channel conformation to the native fold by kinetic and thermodynamic remodelling of the gating energetics towards that of wild-type CTFR.

Rattlesnake Phospholipase A2 Increases CFTR-Chloride Channel Current and Corrects ∆F508CFTR Dysfunction: Impact in Cystic Fibrosis.

Deletion of Phe508 in the nucleotide binding domain (∆F508-NBD1) of the cystic fibrosis transmembrane regulator (CFTR; a cyclic AMP-regulated chloride channel) is the most frequent mutation associated with cystic fibrosis. This mutation affects the maturation and gating of CFTR protein. The search for new high-affinity ligands of CFTR acting as dual modulators (correctors/activators) presents a major challenge in the pharmacology of cystic fibrosis. Snake venoms are a rich source of natural multifunctional proteins, potential binders of ion channels. In this study, we identified the CB subunit of crotoxin from Crotalus durissus terrificus as a new ligand and allosteric modulator of CFTR. We showed that CB interacts with NBD1 of both wild type and ∆F508CFTR and increases their chloride channel currents. The potentiating effect of CB on CFTR activity was demonstrated using electrophysiological techniques in Xenopus laevis oocytes, in CFTR-HeLa cells, and ex vivo in mouse colon tissue. The correcting effect of CB was shown by functional rescue of CFTR activity after 24-h ΔF508CFTR treatments with CB. Moreover, the presence of fully glycosylated CFTR was observed. Molecular docking allowed us to propose a model of the complex involving of the ABCβ and F1-like ATP-binding subdomains of ΔF508-NBD1. Hydrogen-deuterium exchange analysis confirmed stabilization in these regions, also showing allosteric stabilization in two other distal regions. Surface plasmon resonance competition studies showed that CB disrupts the ∆F508CFTR-cytokeratin 8 complex, allowing for the escape of ∆F508CFTR from degradation. Therefore CB, as a dual modulator of ΔF508CFTR, constitutes a template for the development of new anti-CF agents.

New insights into interactions between the nucleotide‐binding domain of CFTR and keratin 8

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide‐binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508‐CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX‐MS) on recombinant wild‐type (wt) NBD1 and ΔF508‐NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt‐NBD1 and ΔF508‐NBD1. Finally, we performed HDX‐MS analysis of the NBD1 molecules and full‐length K8, revealing hydrogen‐bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508‐NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.

Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator

ABSTRACT Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.

ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.