Multi-targeted antifolates aimed at avoiding drug resistance form covalentclosed inhibitory complexes with human and Escherichia coli thymidylate synthases

Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compou nds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-ch aracterized Escherichia coli enzyme, to resolutions of 2.2-3.0 Angstrom. Th e 2.9 Angstrom crystal structure of a complex of human TS with one of the i nhibitors, the multi-targeted antifolate LY231514, demonstrates that this c ompound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the fi rst liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimi dine-based antifolates in complex with Escherichia coli TS confirm the orie ntation of this class of inhibitors in the active site. Specific interactio ns between the polyglutamyl moiety and a positively charged groove on the e nzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d) pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo b y the cellular enzyme folyl polyglutamate synthetase. (C) 2001 Academic Pre ss.