Huennekens F M
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
Adv Enzyme Regul. 1994;34:397-419. doi: 10.1016/0065-2571(94)90025-6.
Methotrexate (MTX), one of the earliest cancer chemotherapy agents, continues to be used extensively in the treatment of leukemia and a variety of other tumors. The efficacy of this drug results from its facile uptake by cells, rapid polyglutamylation and virtually stoichiometric inhibition of dihydrofolate reductase (DHFR), a key enzyme in cell replication. From the work of a multitude of biochemists, molecular biologists, organic chemists and pharmacologists, much is known about the mode of action of MTX and the mechanisms by which tumors exhibit inherent or acquired resistance to this drug. MTX enters cells primarily by a carrier-mediated active transport system whose principal substrate is 5-methyltetrahydrofolate, and additional glutamates are added to the gamma-position of the parent glutamate moiety. The tight binding of MTX to DHFR is defined from NMR and X-ray crystallographic studies of the enzyme and its drug or substrate complexes, supplemented by site-directed mutagenesis to confirm specific interactions. Resistance to the drug, encountered in cell culture model systems or in cancer patients, can result from an increased level of DHFR (due to gene amplification), mutant DHFR with reduced affinity for MTX, or decreased uptake or polyglutamylation of the drug. Although DHFR is an extremely well-studied enzyme, there is still some uncertainty about its kinetics, mechanism for reduction of folate, multiple forms, and activation by a diverse group of agents. Prodrug forms of MTX, e.g., MTX alpha-phenylalanine, which can be activated by carboxypeptidase A-monoclonal antibody conjugates, offer promise for improved efficacy of the drug by selective targeting to tumors. The large body of information summarized above has aided in the development of other folate antagonists, provides a paradigm for assessing the status of other cancer chemotherapeutic agents in current use, and offers a platform from which to speculate about the future of the field.
甲氨蝶呤（MTX）是最早的癌症化疗药物之一，目前仍广泛用于治疗白血病和多种其他肿瘤。该药物的疗效源于其易于被细胞摄取、快速多聚谷氨酸化以及对二氢叶酸还原酶（DHFR）的几乎化学计量的抑制作用，DHFR是细胞复制中的关键酶。通过众多生物化学家、分子生物学家、有机化学家和药理学家的研究，人们对MTX的作用方式以及肿瘤对该药物表现出固有或获得性耐药的机制有了很多了解。MTX主要通过载体介导的主动转运系统进入细胞，其主要底物是5-甲基四氢叶酸，并且在母体谷氨酸部分的γ位添加额外的谷氨酸。MTX与DHFR的紧密结合是通过对该酶及其药物或底物复合物的核磁共振（NMR）和X射线晶体学研究确定的，并辅以定点诱变来确认特定的相互作用。在细胞培养模型系统或癌症患者中遇到的对该药物的耐药性可能源于DHFR水平的升高（由于基因扩增）、对MTX亲和力降低的突变型DHFR，或药物摄取或多聚谷氨酸化的减少。尽管DHFR是一种研究得非常透彻的酶，但其动力学、叶酸还原机制、多种形式以及被多种试剂激活等方面仍存在一些不确定性。MTX的前药形式，例如可被羧肽酶A-单克隆抗体缀合物激活的MTXα-苯丙氨酸，有望通过选择性靶向肿瘤来提高药物疗效。上述大量信息有助于开发其他叶酸拮抗剂，为评估当前使用的其他癌症化疗药物的状况提供了范例，并为推测该领域的未来提供了一个平台。
