Shih C, Chen V J, Gossett L S, Gates S B, MacKellar W C, Habeck L L, Shackelford K A, Mendelsohn L G, Soose D J, Patel V F, Andis S L, Bewley J R, Rayl E A, Moroson B A, Beardsley G P, Kohler W, Ratnam M, Schultz R M
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Cancer Res. 1997 Mar 15;57(6):1116-23.
N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[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 (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) 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 formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.
N-[4-[2-(2-氨基-3,4-二氢-4-氧代-7H-吡咯并[2,3-d]嘧啶-5-基)乙基]-苯甲酰基]-L-谷氨酸(LY231514)是一种新型的基于吡咯并[2,3-d]嘧啶的抗叶酸剂，目前正在进行广泛的II期临床试验。先前的研究已证实，LY231514及其合成的γ-聚谷氨酸(γ-谷氨酰胺三肽和γ-谷氨酰胺五肽)对胸苷酸合酶(TS)具有强效抑制作用。我们现在报告，LY231514及其聚谷氨酸对其他关键的叶酸依赖性酶也有显著抑制作用，包括二氢叶酸还原酶(DHFR)和甘氨酰胺核苷酸甲酰基转移酶(GARFT)。例如，LY231514五聚谷氨酸对TS、DHFR和GARFT抑制作用的Ki值分别为1.3、7.2和65 nM。相比之下，虽然母体单谷氨酸对DHFR(7.0 nM)有类似的高抑制效力，但母体单谷氨酸对TS(109 nM)和GARFT(9300 nM)的抑制常数(Ki)明显较弱。还评估了LY231514及其聚谷氨酸对氨基咪唑甲酰胺核苷酸甲酰基转移酶、5,10-亚甲基四氢叶酸脱氢酶和10-甲酰基四氢叶酸合成酶的影响。在人细胞系中进行的终产物逆转研究进一步支持了细胞毒性涉及多种酶抑制机制的概念。LY231514的逆转模式表明，虽然在接近IC50的浓度下TS可能是LY231514的主要作用位点，但更高的浓度会导致对DHFR和/或嘌呤从头合成途径中的其他酶的抑制。对突变细胞系的研究表明，LY231514需要聚谷氨酸化并通过还原型叶酸载体转运才能发挥细胞毒性作用。因此，我们的数据表明，LY231514是一种新型的经典抗叶酸剂，其抗肿瘤活性可能源于其聚谷氨酸化代谢产物对几种关键的叶酸依赖性酶的同时和多重抑制。
