Human Thymidylate Synthase Is in the Closed Conformation When Complexed with dUMP and Raltitrexed, an Antifolate Drug^,

Abstract

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries. The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution. In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits. This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme. The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A. The thiophene ring of the drug is disordered between two parallel positions.

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

“…The Ca RMSD values between the liganded and unliganded mTS structures presented here, as well as between these structures and the crystal structures of other ternary complexes with dUMP and Tomudex, including those with hTS [15,16], rTS [17] and mTS [18], are shown in Table 3. The superimposition of the mTS, mTS-dUMP and mTS-FdUMP-meTHF structures is shown in Fig.…”

Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

“…Some of the 74 different replacements (or combinations thereof) are responsible for 5-FdUR resistance, including the substitutions in single mutants, whereas other replacements are presumably co-selected and not relevant to resistance. Recent crystallographic analyses of TS, especially closed ternary complexes with dUMP and folate-based inhibitors (14,16), allow us to evaluate these replacements with a view toward understanding possible structure-function relationships and establishing fresh targets for directed mutagenesis. We have used the atomic coordinates of the wild-type amino acids in tightly closed complexes with dUMP and a folate analog inhibitor (see Refs.…”

“…This region has not been reported to be involved in 5-FdUR resistance; structural analysis indicates that it is located on the active site pocket and interacts with folate-based inhibitors, providing a plausible rationale for resistance (1, 11-16). The N-terminal residues from Gly 5 through Gln 18 accumulated 10 substitutions; this segment is disordered in existing crystal structures and has no known function (11)(12)(13)(14)(15)(16). There is no obvious correlation between the frequency of either single or multiple substitutions observed in resistant mutants and the presence of ␤-sheets or ␣-helices.…”

“…Although metabolically unstable, the P303L protein was nonetheless able to confer resistance against both 5-FdUR-and anti-folate-based TS inhibitors in transfected cells (10). Like Y33H, the P303L substitution is not in close proximity to the catalytic site as determined from crystal structures (11)(12)(13)(14)(15)(16).…”

Molecular Docking Studies of Phytoconstituents Identified in Crocus sativus, Curcuma longa, Cassia occidentalis and Moringa oleifera on Thymidylate Synthase and ndash; An Enzyme Target for Anti-Cancer Activity

“…Docking analysis is done by initially selecting the target for the disease and followed by obtaining the 3D structure of thymidylate syntahse (1HVY) (Phan et al, 2001) from protein data bank (Bernstein et al, 1978) in .pdb format. It is well known that PDB files often have poor or missing assignments of explicit hydrogens, and the PDB file format cannot accommodate bond order information.…”