LY231514, A PYRROLO[2,3-D]PYRIMIDINE-BASED ANTIFOLATE THAT INHIBITS MULTIPLE FOLATE-REQUIRING ENZYMES

lo[2,3-d]pyrimidin-5-yl)ethyl]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu(3) and glu( 5)) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit othe r key folate-requiring enzymes, including dihydrofolate reductase (DHF R) and glycinamide ribonucleotide formyltransferase (GARFT). For examp le, the K-i values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In co ntrast, although a similar high level of inhibitory potency was observ ed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (K-i) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltran sferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formylte trahydrofolate synthetase were also evaluated. The end product reversa l studies conducted in human cell lines further support the concept th at multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a m ajor site of action for LY231514 at concentrations near the IC50, high er concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demon strated that LY231514 requires polyglutamation and transport via the r educed folate carrier for cytotoxic potency. Therefore, our data sugge st that LY231514 is a novel classical antifolate, the antitumor activi ty of which may result from simultaneous and multiple inhibition of se veral key folate-requiring enzymes via its polyglutamated metabolites.