Binding of the anticancer drug ZD1694 to E. coli thymidylate synthase: assessing specificity and affinity.
BACKGROUND
Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by 5, 10-methylenetetrahydrofolate (CH2H4folate) to form deoxythymidine monophosphate (dTMP) and dihydrofolate (H2folate). The essential role of TS in the cell life cycle makes it an attractive target for the development of substrate and cofactor-based inhibitors that may find efficacy as anticancer and antiproliferative drugs. Antifolates that compete specifically with the binding of CH2H4 folate include the cofactor analog CB3717 (10-propargyl-5,8-dideazafolate). However, the development of potent cofactor analog inhibitors of TS, such as CB3717, as drugs has been slowed by their toxicity, which often becomes apparent as hepatic and renal toxicity mediated by the specific chemistry of the compound. Attempts to abolish toxicity in human patients while preserving potency against the target enzyme, have led to the development of ZD1694, which has already shown significant activity against colorectal tumours.


RESULTS
The three dimensional crystallographic structure of ZD1694 in complex with dUMP and Escherichia coli TS has been determined to a resolution of 2.2  . This was used to evaluate the specific structural determinants of ZD1694 potency and to correlate structure/activity relationships between it and the closely related ligand, CB3717. ZD1694 binds to TS in the same manner as CB3717 and H2 folate, but a methyl group on its quinazoline ring, its thiophene ring and the methyl group at N10 are compensated for by plastic accommodation of the enzyme active site coupled with specific rearrangement in the solvent structure. A specific hydrogen bond between the protein and the inhibitor CB3717 is absent in the case of ZD1694 whose monoglutamate tail is reoriented and more well ordered.


CONCLUSIONS
The binding mode of ZD1694 to thymidylate synthase has been determined at atomic resolution. ZD1694 forms a ternary complex with dUMP and participates in the multi-step TS reaction through the covalent bond formation between dUMP and Cys146 thereby competing with CH2H4 folate at the active site. Analysis of this inhibitor ternary complex structure and comparison with that of CB3717 reveals that the enzyme accommodates the differences between the two inhibitors with small shifts in the positions of key active site residues and by repositioning an active site water molecule, thereby preserving a general binding mode of these inhibitors.
Rutenber
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Department of Biochemistry and Biophysics, University of California at San Francisco, 94143-0448, USA. stroud@msg.ucsf.edu
Stroud
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D000964:Antimetabolites, Antineoplastic / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011799:Quinazolines / Q000737:chemistry* / Q000378:metabolism; D013045:Species Specificity; D013876:Thiophenes / Q000737:chemistry* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D005493:Folic Acid Antagonists; D011799:Quinazolines; D013876:Thiophenes; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase; C068874:raltitrexed