A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase.
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.
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, UK.
Hugh P
HP
Morgan
Martin J
MJ
Walsh
Elizabeth A
EA
Blackburn
Martin A
MA
Wear
Matthew B
MB
Boxer
Min
M
Shen
Henrike
H
Veith
Iain W
IW
McNae
Matthew W
MW
Nowicki
Paul A M
PA
Michels
Douglas S
DS
Auld
Linda A
LA
Fothergill-Gilmore
Malcolm D
MD
Walkinshaw
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001565:Benzoates / Q000494:pharmacology; D020134:Catalytic Domain / Q000187:drug effects; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007894:Leishmania mexicana / Q000201:enzymology; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012439:Saccharin / Q000031:analogs & derivatives / Q000494:pharmacology; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013498:Suramin / Q000494:pharmacology
C577329:4-((1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl)benzoic acid; D001565:Benzoates; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D013498:Suramin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D011770:Pyruvate Kinase; D012439:Saccharin; D008239:Lysine