raltitrexed and 2--deoxyuridylic-acid

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 A and 2.5 A resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 A away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten-fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

Topics: Binding Sites; Catalytic Domain; Crystallography, X-Ray; Cysteine; Deoxyuracil Nucleotides; Drug Design; Enzyme Inhibitors; Escherichia coli; Humans; Models, Molecular; Protein Binding; Protein Structure, Secondary; Quinazolines; Sequence Deletion; Structure-Activity Relationship; Thiophenes; Thymidylate Synthase

Two crystal structures of rat thymidylate synthase (TS) complexed with dUMP and the anticancer drug Tomudex (ZD1694) have been determined to resolutions of 3.3 and 2.6 A. Tomudex is one of several new antifolates targeted to TS and the first to be approved for clinical use. The structures represent the first views of any mammalian TS bound to ligands and suggest that the rat protein undergoes a ligand-induced conformational change similar to that of the Escherichia coli protein. Surprisingly, Tomudex does not induce the "closed" conformation in rat TS that is seen on binding to E. coli TS, resulting in inhibitor atoms that differ in position by more than 1.5 A. Several species-specific differences in sequence may be the reason for this. Phe 74 shifts to a new position in the rat complex and is in van der Waals contact with the inhibitor, while in the E. coli protein the equivalent amino acid (His 51) hydrogen bonds to the glutamate portion of the inhibitor. Amino acids Arg 101, Asn 106, and Met 305 make no contacts with the inhibitor in the open conformation, unlike the equivalent residues in the E. coli protein (Thr 78, Trp 83, and Val 262). dUMP binding is similar in both proteins, except that there is no covalent adduct to the active site cysteine (Cys 189) in the rat structures. Two insertions in the rat protein are clearly seen, but the N-termini (residues 1-20) and C-termini (residues 301-307) are disordered in both crystal forms.

Topics: Animals; Antimetabolites, Antineoplastic; Bacterial Proteins; Carcinoma, Hepatocellular; Computer Simulation; Crystallography, X-Ray; Deoxyuracil Nucleotides; Humans; Ligands; Models, Molecular; Protein Conformation; Quinazolines; Rats; Recombinant Proteins; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured