HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end com-plex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406–415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.
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Research Article|
September 27 2006
Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design
Kamel EL Omari;
Kamel EL Omari
*Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, U.K.
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Annelies Bronckaers;
Annelies Bronckaers
†Rega Institute for Medical Research, K.U.Leuven, B-3000 Leuven, Belgium
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Sandra Liekens;
Sandra Liekens
†Rega Institute for Medical Research, K.U.Leuven, B-3000 Leuven, Belgium
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Maria-Jésus Pérez-Pérez;
Maria-Jésus Pérez-Pérez
‡Instituto de Química Médica (C.S.I.C.), 28006 Madrid, Spain
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Jan Balzarini;
Jan Balzarini
†Rega Institute for Medical Research, K.U.Leuven, B-3000 Leuven, Belgium
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David K. Stammers
David K. Stammers
1
*Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, U.K.
1To whom correspondence should be addressed (email daves@strubi.ox.ac.uk).
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Publisher: Portland Press Ltd
Received:
April 06 2006
Revision Received:
June 21 2006
Accepted:
June 28 2006
Accepted Manuscript online:
June 28 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 399 (2): 199–204.
Article history
Received:
April 06 2006
Revision Received:
June 21 2006
Accepted:
June 28 2006
Accepted Manuscript online:
June 28 2006
Citation
Kamel EL Omari, Annelies Bronckaers, Sandra Liekens, Maria-Jésus Pérez-Pérez, Jan Balzarini, David K. Stammers; Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design. Biochem J 15 October 2006; 399 (2): 199–204. doi: https://doi.org/10.1042/BJ20060513
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