Glutamyl hydrolase and the multitargeted antifolate LY231514

Purpose: To examine the activity of glutamyl hydrolase (GH) on the poly-gam ma-glutamates of multitargeted antifolate (MTA) (LY231514) and the effect o f enhanced GH on the pharmacological activity of MTA. Methods: Expressed an d purified GH were used to study the enzymatic cleavage of MTA poly-gamma-g lutamates and wild-type and GH-enhanced H35 hepatoma cell lines to evaluate growth inhibition. Results: MTA tri- and penta-gamma-glutamates were good substrates for human GH, having higher rates than MTX tri- and penta-gamma- glutamates. Preferential hydrolysis with human enzyme occurred at the two g amma-glutamyl bonds at the carboxyl end of the molecule, whereas the rat en zyme preferred the innermost gamma-linkage. Incubation of rat H35 hepatoma cell lines with MTA resulted in the intracellular accumulation of primarily tetra-, penta-, and hexa-gamma-glutamate. The formation of these were mark edly reduced in H35D cells, which is a line resistant to antifolates chiefl y through enhanced cellular levels of GH activity. Conclusions: MTA poly-ga mma-glutamates are effective substrates for GH and their pharmacological ef fectiveness bears an inverse relationship to cellular GH activity. This obs ervation, along with enhanced resistance to MTA of thymidylate synthase-amp lified cells, substantiates the importance of the poly-gamma-glutamates of MTA inhibiting TS as the primary target. Further evidence for the inverse r elationship of GH to classical antifolate pharmacological activity is estab lished.