The role of phosphate in the action of thymidine phosphorylase inhibitors: Implications for the catalytic mechanism

• Published in: Bioorganic & medicinal chemistry letters 2010-03, Vol.20 (5), p.1648-1651
• Main Authors: Jain, Harsh V., Rasheed, Roshni, Kalman, Thomas I.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Catalytic Domain; Enzyme Inhibitors - chemistry; Enzyme mechanism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Inhibition kinetics; Kinetics; Phosphates - chemistry; Protein Binding; Static Electricity; Thymidine phosphorylase; Thymidine Phosphorylase - antagonists & inhibitors; Thymidine Phosphorylase - metabolism; Thymine - analogs & derivatives; Thymine - chemistry; Thymine - pharmacology; Transferases; Transition state analog
• ISSN: 0960-894X; 1464-3405
• DOI: 10.1016/j.bmcl.2010.01.076

The inhibition of thymidine phosphorylase by AIFU was determined to follow uncompetitive kinetics with respect to inorganic phosphate, indicating that zwitterionic transition state analogs bind to the enzyme phosphate binary complex, consistent with an S N2-type catalytic mechanism. The design and synthesis of 5-fluoro-6-[(2-aminoimidazol-1-yl)methyl]uracil (AIFU), a potent inhibitor of thymidine phosphorylase (TP) with K i-values of 11 nM (ecTP) and 17 nM (hTP), are described. Kinetic studies established that the type of inhibition of TP by AIFU is uncompetitive with respect to inorganic phosphate (or arsenate). The results obtained suggest that AIFU and other zwitterionic thymine analog inhibitors of TP act as transition state analogs, mimicking the anionic thymine leaving group, consistent with an S N2-type catalytic mechanism, and anchored by their protonated side chains to the enzyme-bound phosphate by electrostatic and H-bonding interactions.