Enzyme inhibition, polyglutamation, and the effect of LY231514 (MTA) on purine biosynthesis.
The pyrrolopyrimidine-based antifolate, N-¿4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]benzoyl¿glutamic acid, LY231514 (MTA) has demonstrated antitumor activity in a broad array of human tumors, including breast cancer, colon cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, and other solid tumors. The biochemical basis of this activity was explored by measuring activation of MTA by polyglutamation and the activity of MTA to inhibit several folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). The enzyme folylpolyglutamate synthase (FPGS) activated MTA very efficiently. Using FPGS from two different species, Km values below 2 micromol/L and high relative first order rate constants, k' (Vmax/Km) of 6.4 and 13.7 compared with another substrate, lometrexol, were obtained. The formation of polyglutamates of several antifolates were compared in vitro at high and low substrate concentrations. At low MTA concentrations, tetraglutamated and pentaglutamated MTA were the predominant forms identified after a 24-hour incubation period. In contrast, only diglutamyl methotrexate and a mixture triglutamylated, tetraglutamylated, and pentaglutamylated forms of the GARFT inhibitor lometrexol were formed under the same conditions. At higher substrate concentrations (20 micromol/L, 24 hours), greater amounts of each product were formed. The major metabolites, however, were triglutamated MTA or triglutamated lometrexol, while only diglutamyl methotrexate was recovered. Thus, MTA was an excellent substrate for FPGS and it was efficiently metabolized to highly polyglutamated species by this enzyme. The activity of MTA and its polyglutamated metabolites to inhibit several folate-dependent enzymes was measured. In vitro, MTA and its polyglutamates were potent, tight-binding inhibitors of several folate-dependent enzymes, including thymidylate synthase, dihydrofolate reductase, and GARFT. Preliminary cell-based assays (CCRF-CEM) demonstrated inhibition of the purine de novo pathway by MTA, consistent with its multitargeted mechanism of action against tumor cells. The combined effects of activation of MTA to highly polyglutamated metabolites and the potency of these polyglutamates to inhibit multiple folate-dependent enzymes provide a mechanistic basis for understanding the broad antitumor activity of this compound against many human tumor types.
Mendelsohn
L G
LG
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis 46285, USA.
Shih
C
C
Chen
V J
VJ
Habeck
L L
LL
Gates
S B
SB
Shackelford
K A
KA
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology; D000068437:Pemetrexed; D010453:Peptide Synthases / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000378:metabolism; D011687:Purines / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013763:Tetrahydrofolates / Q000378:metabolism / Q000494:pharmacology
D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D005971:Glutamates; D011687:Purines; D013763:Tetrahydrofolates; D000068437:Pemetrexed; D011099:Polyglutamic Acid; D006147:Guanine; C045894:lometrexol; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; C030985:purine; D008727:Methotrexate