Purpose: To examine the activity of glutamyl hydrolase (GH) on the poly-gamma-glutamates of multitargeted antifolate (MTA) (LY231514) and the effect of enhanced GH on the pharmacological activity of MTA.
Methods: Expressed and purified GH were used to study the enzymatic cleavage of MTA poly-gamma-glutamates 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 gamma-glutamyl bonds at the carboxyl end of the molecule, whereas the rat enzyme 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 markedly reduced in H35D cells, which is a line resistant to antifolates chiefly through enhanced cellular levels of GH activity.
Conclusions: MTA poly-gamma-glutamates are effective substrates for GH and their pharmacological effectiveness bears an inverse relationship to cellular GH activity. This observation, along with enhanced resistance to MTA of thymidylate synthase-amplified cells, substantiates the importance of the poly-gamma-glutamates of MTA inhibiting TS as the primary target. Further evidence for the inverse relationship of GH to classical antifolate pharmacological activity is established.