Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase

The mechanism of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase was investigated. A functionally reactive chloroacetyl group was substituted for the propargyl group to examine whether the resulting analogue of CB3717 could become an irreversible inhibitor of thymidylate synthase. Results show that a sulfhydryl reactive residue directed to the folate binding site of thymidylate synthase may diffuse to the active site cysteine and form a covalent bond with the compound.