Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidine-based antifolate LY231514 (MTA)
Introduction
Since the late 1950 s, extensive research efforts have been devoted to the discovery and development of antifolate antimetabolites as chemotherapeutic agents for the management of neoplastic diseases. However, it was in the last 10 to 15 years, due to the rapid advances of medicinal chemistry, x-ray protein crystallography, molecular biology, pharmacology and clinical medicine, that a significant number of new generation antifolates were brought forward for clinical development. Several folate-based antimetabolites are currently being investigated in clinical trials. These include Lometrexol (6R-5,10-dideazatetrahydrofolic acid, DDATHF) (1-3), LY309887[4]and AG2034[5], which exhibit potent and selective inhibition of glycinamide ribonucleotide formyltransferase (GARFT, EC 2.1.2.2) of the purine de novo biosynthetic pathway; trimetrexate[6], edatrexate7, 8and PT523[9]which act on dihydrofolate reductase (DHFR, EC 1.5.1.3); Tomudex®10, 11, AG337[12], BW1843U89[13]and ZD9331[14]which target specifically at the enzyme thymidylate synthase (TS, EC 2.1.1.45) of the pyrimidine biosynthesis.
N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]-benzoyl]-L-glutamic acid, LY231514 (Multitargeted Antifolate, MTA), is a structurally novel antifolate antimetabolite that possesses the unique 6-5 fused pyrrolo[2,3-d]pyrimidine nucleus instead of the more common 6-6 fused pteridine or quinazoline ring structure[15](Fig. 1). As a “classical” antifolate, LY231514 was found to be one of the best substrates for the mammalian folylpolyglutamate synthetase (FPGS, EC 6.3.2.17) and it is believed that polyglutamation and the polyglutamated metabolites of LY231514 play profound roles in determining both the selectivity and the antitumor activity of this novel agent15, 16. Preliminary cell culture end-product reversal studies in human CCRF-CEM and murine L1210 leukemia cells have demonstrated that thymidine (5 μM) alone was not able to fully reverse the cytotoxic action of LY231514. Both thymidine (5 μM) and hypoxanthine (100 μM) were required to fully protect cells from the growth inhibitory activity exerted by LY231514[15]. This reversal pattern is significantly different from other known antifolates such as methotrexate, Tomudex® and the GARFT inhibitor DDATHF, and suggests that TS is only partially responsible for the antiproliferative action of this novel antifolate. LY231514 and its polyglutamates may inhibit other folate-requiring enzymes, such as DHFR, or enzymes along the de novo purine biosynthetic pathway. This report summarizes our findings on the polyglutamation profile of LY231514, the activity of LY231514-polyglutamates (glu3 and glu5) against various folate-requiring enzymes, the cell culture reversal and cross-resistance pattern of LY231514 and, finally, the effects of LY231514 on cellular folate and nucleoside triphosphate pools.
Section snippets
Materials
Methotrexate, hypoxanthine and thymidine were obtained from Sigma Chemical Company, St. Louis, MO. Methotrexate polyglutamates were purchased from Dr. Schirks Laboratories, Jona, Switzerland. LY231514 and Lometrexol were prepared at Lilly Research Laboratories, Indianapolis, IN. The syntheses of the γ-glutamyl derivatives of LY231514 were by the method of Pawelczak et al.[17]. L-[14C(U)]-glutamate (NEC-290E) was acquired from Dupont NEN, Boston, MA. The Enzfitter microcomputer package was
Polyglutamation profile of LY231514
Polyglutamation plays an essential role in determining the overall biochemical and pharmacological properties of the “classical” antifolates. The formation of polyglutamated metabolites of folates and antifolates results in the intracellular accumulation of polyglutamated metabolites to levels that are significantly higher than could otherwise be achieved at steady state by the parent compounds, and thus serves as an important cellular retention mechanism for folates and antifolates26, 27. The
Summary
Extensive biochemical and pharmacological evidence indicates that LY231514 is a novel antifolate antimetabolite. LY231514 is transported into cells mainly through the reduced folate carrier system and extensively metabolized to polyglutamated forms. The polyglutamates of LY231514 inhibit at least three key folate enzymes: TS, DHFR, and GARFT, and to a lesser extent AICARFT and C1-tetrahydrofolate synthase. The combined effects of the inhibition exerted by LY231514 at each target give rise to an
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