McGuire J J, Hart B P, Haile W H, Rhee M S, Galivan J, Coward J K
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Arch Biochem Biophys. 1995 Aug 20;321(2):319-28. doi: 10.1006/abbi.1995.1401.
Poly(gamma-glutamylation) of glutamate (L-Glu)-containing antifolates and natural folates is important in pharmacological mechanisms and in physiological processes. Based on previous work from our laboratories, we hypothesized that replacement of the L-Glu moiety in parent molecules with DL-beta,beta-difluoroglutamic acid (DL-beta,beta-F2Glu) might be a generic means of increasing polyglutamylation by both increasing the synthesis rate and decreasing the degradation rate (J. J. McGuire et al., J. Biol. Chem. 265, 14073-14079 (1990)); thus biological potency might be increased without other biochemical properties being altered. DL-beta,beta-F2Glu, synthesized by an improved route (B. P. Hart and J. K. Coward, Tetrahedron Lett. 34, 4917-4920 (1993)), has been incorporated into a methotrexate (MTX) homolog, beta,beta-difluoromethotrexate (beta,beta-F2MTX), and a folic acid (PteGlu) homolog, beta,beta-difluorofolic acid (beta,beta-PteF2Glu). Biochemical properties of beta,beta-F2MTX (e.g., inhibition of isolated dihydrofolate reductase, transport in whole cells) are similar to those of MTX except that, in accord with our hypothesis, apparent substrate efficiency for rat and human folylpolyglutamate synthetase (FPGS) is 4- to 7.5-fold higher, respectively, for beta,beta-F2MTX than for MTX. Analysis of the products synthesized by purified FPGS, however, suggests that while addition of the first gamma-Glu to beta,beta-F2MTX is highly efficient, subsequent additions occur at a negligible rate; this premise was confirmed by directly comparing the in vitro FPGS substrate activity of MTX-gamma-Glu and beta,beta-F2MTX-gamma-Glu. Furthermore, the dramatically diminished in vitro growth inhibitory potency of beta,beta-F2MTX as compared to MTX when exposure time to drug is decreased (despite otherwise similar biochemical properties) suggests that polyglutamylation is also impaired in intact cells. Similar results with FPGS have been obtained with oxidized and reduced forms of beta,beta-PteF2Glu. These data suggest that the effect of beta,beta-F2Glu on polyglutamylation by FPGS is dependent on its position relative to the point of L-Glu ligation. When beta,beta-F2Glu is the acceptor amino acid (i.e., point of attachment), ligation of Glu is enhanced; however, if beta,beta-F2Glu is one residue distal to the acceptor amino acid, further elongation is blocked.
含谷氨酸（L-Glu）的抗叶酸药物和天然叶酸的聚（γ-谷氨酰化）在药理机制和生理过程中具有重要意义。基于我们实验室之前的工作，我们推测用DL-β,β-二氟谷氨酸（DL-β,β-F2Glu）取代母体分子中的L-Glu部分可能是一种通用方法，通过提高合成速率和降低降解速率来增加聚谷氨酰化（J. J. McGuire等人，《生物化学杂志》265, 14073 - 14079 (1990)）；因此，在不改变其他生化特性的情况下，生物活性可能会增加。通过改进路线合成的DL-β,β-F2Glu（B. P. Hart和J. K. Coward，《四面体快报》34, 4917 - 4920 (1993)）已被并入甲氨蝶呤（MTX）类似物β,β-二氟甲氨蝶呤（β,β-F2MTX）和叶酸（PteGlu）类似物β,β-二氟叶酸（β,β-PteF2Glu）中。β,β-F2MTX的生化特性（例如，对分离的二氢叶酸还原酶的抑制作用、在全细胞中的转运）与MTX相似，只是根据我们的假设，β,β-F2MTX对大鼠和人类叶酸聚谷氨酰胺合成酶（FPGS）的表观底物效率分别比MTX高4至7.5倍。然而，对纯化的FPGS合成产物的分析表明，虽然将第一个γ-Glu添加到β,β-F2MTX上效率很高，但随后的添加速率可以忽略不计；通过直接比较MTX-γ-Glu和β,β-F2MTX-γ-Glu的体外FPGS底物活性，这一前提得到了证实。此外，当药物暴露时间减少时（尽管其他生化特性相似），与MTX相比，β,β-F2MTX的体外生长抑制活性显著降低，这表明在完整细胞中聚谷氨酰化也受到损害。对于β,β-PteF2Glu的氧化形式和还原形式，使用FPGS也得到了类似的结果。这些数据表明，β,β-F2Glu对FPGS介导的聚谷氨酰化的影响取决于其相对于L-Glu连接点的位置。当β,β-F2Glu是受体氨基酸（即连接点）时，Glu的连接增强；然而，如果β,β-F2Glu是受体氨基酸远端的一个残基，则进一步的延伸被阻断。
