Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease

2009 ◽  
Vol 418 (1) ◽  
pp. 185-190 ◽  
Author(s):  
Stan Pasyk ◽  
Canhui Li ◽  
Mohabir Ramjeesingh ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Small Molecule ◽  
Mechanism Of Action ◽  
Severe Disease ◽  
Atp Binding ◽  
Molecular Defect ◽  
Apical Surface ◽  
Channel Activity ◽  
Small Molecule Modulator

CF (cystic fibrosis) is caused by mutations in CFTR (CF transmembrane conductance regulator), which cause its mistrafficking and/or dysfunction as a regulated chloride channel on the apical surface of epithelia. CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. However, the molecular target(s) for these modulators and their mechanism of action remain unclear. In the present study, we evaluated the mechanism of action of one small-molecule modulator, VRT-532, identified as a specific modulator of CF-causing mutants. First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Interestingly, VRT-532 did not affect the ATPase activity of the Wt (wild-type) CFTR, supporting the idea that this compound corrects the specific molecular defect in this mutant. To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis.

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

2008 ◽  
Vol 412 (2) ◽  
pp. 315-321 ◽  
Author(s):  
Mohabir Ramjeesingh ◽  
Francisca Ugwu ◽  
Fiona L. L. Stratford ◽  
Ling-Jun Huan ◽  
Canhui Li ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Mechanism Of Action ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Residual Activity ◽  
Atp Binding ◽  
Cftr Protein ◽  
The Individual ◽  
Atp Binding And Hydrolysis

The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain–domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy

2011 ◽  
Vol 50 ◽  
pp. 233-248 ◽  
Author(s):  
Patrick Kim Chiaw ◽  
Paul D.W. Eckford ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecule ◽  
Molecular Basis ◽  
Structural Changes ◽  
Common Mutation ◽  
Channel Activity ◽  
Wild Type ◽  
In The Wild ◽  
Cftr Protein ◽  
Small Molecule Modulators

Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway obstruction with recurrent airway inflammation and infection. Pulmonary obstruction in CF has been linked to the loss of CFTR function as a regulated Cl− channel on the lumen-facing membrane of the epithelium lining the airways. We have learned much about the molecular basis for nucleotide- and phosphorylation-dependent regulation of channel activity of the normal (wild-type) version of the CFTR protein through electrophysiological studies. The major CF-causing mutation, F508del-CFTR, causes the protein to misfold and be retained in the ER (endoplasmic reticulum). Importantly, recent studies in cell culture have shown that retention in the ER can be ‘corrected’ through the application of certain small-molecule modulators and, once at the surface, the altered channel function of the major mutant can be ‘potentiated’, pharmacologically. Importantly, two such small molecules, a ‘corrector’ (VX-809) and a ‘potentiator’ (VX-770) compound are undergoing clinical trial for the treatment of CF. In this chapter, we describe recent discoveries regarding the wild-type CFTR and F508del-CFTR protein, in the context of molecular models based on X-ray structures of prokaryotic ABC (ATP-binding cassette) proteins. Finally, we discuss the promise of small-molecule modulators to probe the relationship between structure and function in the wild-type protein, the molecular defects caused by the most common mutation and the structural changes required to correct these defects.

scholarly journals Dysfunctional Inflammation in Cystic Fibrosis Airways: From Mechanisms to Novel Therapeutic Approaches

2021 ◽  
Vol 22 (4) ◽  
pp. 1952
Author(s):  
Alessandra Ghigo ◽  
Giulia Prono ◽  
Elisa Riccardi ◽  
Virginia De Rose
Keyword(s):  
Cystic Fibrosis ◽  
Critical Role ◽  
Airway Epithelial Cells ◽  
Lung Damage ◽  
Molecular Defect ◽  
Apical Surface ◽  
Therapeutic Approaches ◽  
Beneficial Effects ◽  
Anti Inflammatory ◽  
Novel Therapeutic

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.

A Topological Switch in the Cystic Fibrosis Transmembrane Conductance Regulator Modulates Channel Activity and Sensitivity to Disease-Causing Mutation

2020 ◽  
Author(s):  
Daniel Scholl ◽  
Maud Sigoillot ◽  
Marie Overtus ◽  
Rafael Colomer ◽  
Chloé Martens ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Single Molecule ◽  
Genetic Disorder ◽  
Anion Channel ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs such as the lungs or the digestive systems. Functional impairment of CFTR due to mutation in the cftr gene lead to Cystic Fibrosis (CF) the most common lethal genetic disorder. Here we observe that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and other ATP-binding cassette (ABC) transporter proteins. Crystallography studies reveal that this conformation involves a topological reorganization of the β-subdomain of NBD1. This alternative state is adopted by wild-type CFTR in cells, where it leads to enhanced channel activity. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. However, under destabilizing conditions, such as a the prominent disease-causing mutation F508el , this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that in wild-type CFTR switching to this topologically-swapped conformation of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

scholarly journals Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain

2000 ◽  
Vol 116 (2) ◽  
pp. 163-180 ◽  
Author(s):  
Kim W. Chan ◽  
László Csanády ◽  
Donna Seto-Young ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Flag Epitope ◽  
Mature Form ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

The cystic fibrosis transmembrane conductance regulator is a Cl− channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590–830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH2- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH2 terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH2 termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH2-terminal segment representing aa's 3–622 (Flag3-622) or 3–633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634–1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.

A Small-Molecule Modulator Interacts Directly with ΔPhe508-CFTR to Modify Its ATPase Activity and Conformational Stability

2009 ◽  
Vol 75 (6) ◽  
pp. 1430-1438 ◽  
Author(s):  
Leigh Wellhauser ◽  
Patrick Kim Chiaw ◽  
Stan Pasyk ◽  
Canhui Li ◽  
Mohabir Ramjeesingh ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Small Molecule ◽  
Conformational Stability ◽  
Small Molecule Modulator
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