A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

DOI: 10.1042/BJ20121014

Authors: Hugh Morgan (University of Edinburgh) , Martin J. Walsh (National Human Genome Research Institute, National Institutes of Health) , Elizabeth A. Blackburn (University of Edinburgh) , Martin A. Wear (University of Edinburgh) , Matthew B. Boxer (National Human Genome Research Institute, National Institutes of Health) , Min Shen (National Human Genome Research Institute, National Institutes of Health) , Iain W. Mcnae (University of Edinburgh) , Matthew W. Nowicki (University of Edinburgh) , Paul A. M. Michels (Université catholique de Louvain) , Douglas Auld (National Human Genome Research Institute, National Institutes of Health) , Linda Fothergill-Gilmore (University of Edinburgh) , Malcolm D. Walkinshaw (University of Edinburgh) , Henrike Veith (National Human Genome Research Institute, National Institutes of Health)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal

State: Published (Approved)
Published: September 2012
Diamond Proposal Number(s): 7613

Abstract: 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.

Journal Keywords: covalent inhibitor; Leishmania mexicana; lysine covalent modification; nucleotide binding; pyruvate kinase; saccharin analogue

Diamond Keywords: Leishmaniasis

Subject Areas: Biology and Bio-materials, Chemistry, Medicine


Instruments: I03-Macromolecular Crystallography

Added On: 27/09/2012 14:33

Discipline Tags:

Infectious Diseases Disease in the Developing World Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech Parasitology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)