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.
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Research Article|
January 28 2009
Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease
Stan Pasyk;
Stan Pasyk
*Programme in Molecular Structure and Function, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8
†Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5G 1X8
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Canhui Li;
Canhui Li
*Programme in Molecular Structure and Function, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8
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Mohabir Ramjeesingh;
Mohabir Ramjeesingh
*Programme in Molecular Structure and Function, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8
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Christine E. Bear
Christine E. Bear
1
*Programme in Molecular Structure and Function, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8
†Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5G 1X8
‡Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5G 1X8
1To whom correspondence should be addressed (email bear@sickkids.ca).
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Publisher: Portland Press Ltd
Received:
July 14 2008
Revision Received:
September 19 2008
Accepted:
October 23 2008
Accepted Manuscript online:
October 23 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 418 (1): 185–190.
Article history
Received:
July 14 2008
Revision Received:
September 19 2008
Accepted:
October 23 2008
Accepted Manuscript online:
October 23 2008
Citation
Stan Pasyk, Canhui Li, Mohabir Ramjeesingh, Christine E. Bear; Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease. Biochem J 15 February 2009; 418 (1): 185–190. doi: https://doi.org/10.1042/BJ20081424
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