Glutamyl hydrolase and the multitargeted antifolate LY231514.
OBJECTIVE
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.
Rhee
M S
MS
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509, USA.
Ryan
T J
TJ
Galivan
J
J
D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology; D005971:Glutamates / Q000493:pharmacokinetics* / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D000068437:Pemetrexed; D051381:Rats; D013379:Substrate Specificity; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism*
D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D005971:Glutamates; D000068437:Pemetrexed; D006147:Guanine; D011623:gamma-Glutamyl Hydrolase