BINDING OF THE ANTICANCER DRUG ZD1694 TO ESCHERICHIA-COLI THYMIDYLATESYNTHASE - ASSESSING SPECIFICITY AND AFFINITY

Background: Thymidylate synthase (TS) catalyzes the reductive methylat ion of deoxyuridine monophosphate (dUMP) by 5,10-methylenetetrahydrofo late (CH(2)H(4)folate) to form deoxythymidine monophosphate (dTMP) and dihydrofolate (H(2)folate). 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 t he binding of CH2H4 folate include the cofactor analog CB3717 (10-prop argyl-5,8-dideazafolate). However, the development of potent cofactor analog inhibitors of TS, such as CB3717, as drugs has been slowed by t heir toxicity, which often becomes apparent as hepatic and renal toxic ity mediated by the specific chemistry of the compound. Attempts to ab olish toxicity in human patients while preserving potency against the target enzyme, have led to the development of ZD1694, which has alread y 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 Angstrom. This was used to evaluate the specific structural determi nants of ZD1694 potency and to correlate structure/activity relationsh ips between it and the closely related ligand, CB3717. ZD1694 binds to TS in the same manner as C83717 and H(2)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 speci fic hydrogen bond between the protein and the inhibitor CB3717 is abse nt in the case of ZD1694 whose monoglutamate tail is reoriented and mo re well ordered. Conclusions: The binding mode of ZD1694 to thymidylat e synthase has been determined at atomic resolution. ZD1694 forms a te rnary complex with dUMP and participates in the multi-step TS reaction through the covalent bond formation between dUMP and Cys146 thereby c ompeting with CH(2)H(4)folate at the active site. Analysis of this inh ibitor ternary complex structure and comparison with that of CB3717 re veals that the enzyme accommodates the differences between the two inh ibitors with small shifts in the positions of key active site residues and by repositioning an active site water molecule, thereby preservin g a general binding mode of these inhibitors. (C) Current Biology Ltd