PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.
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Research Article|
October 18 2012
A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase
Hugh P. Morgan;
Hugh P. Morgan
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
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Martin J. Walsh;
Martin J. Walsh
†NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
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Elizabeth A. Blackburn;
Elizabeth A. Blackburn
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
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Martin A. Wear;
Martin A. Wear
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
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Matthew B. Boxer;
Matthew B. Boxer
†NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
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Min Shen;
Min Shen
†NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
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Henrike Veith;
Henrike Veith
†NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
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Iain W. McNae;
Iain W. McNae
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
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Matthew W. Nowicki;
Matthew W. Nowicki
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
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Paul A. M. Michels;
Paul A. M. Michels
‡Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Université catholique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium
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Douglas S. Auld;
Douglas S. Auld
†NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, U.S.A.
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Linda A. Fothergill-Gilmore;
Linda A. Fothergill-Gilmore
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
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Malcolm D. Walkinshaw
Malcolm D. Walkinshaw
1
*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
1To whom correspondence should be addressed (email m.walkinshaw@ed.ac.uk).
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Publisher: Portland Press Ltd
Received:
June 25 2012
Revision Received:
July 27 2012
Accepted:
August 20 2012
Accepted Manuscript online:
August 20 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 448 (1): 67–72.
Article history
Received:
June 25 2012
Revision Received:
July 27 2012
Accepted:
August 20 2012
Accepted Manuscript online:
August 20 2012
Citation
Hugh P. Morgan, Martin J. Walsh, Elizabeth A. Blackburn, Martin A. Wear, Matthew B. Boxer, Min Shen, Henrike Veith, Iain W. McNae, Matthew W. Nowicki, Paul A. M. Michels, Douglas S. Auld, Linda A. Fothergill-Gilmore, Malcolm D. Walkinshaw; A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase. Biochem J 15 November 2012; 448 (1): 67–72. doi: https://doi.org/10.1042/BJ20121014
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