Rosowsky A, Wright J E, Shapiro H, Beardsley P, Lazarus H
J Biol Chem. 1982 Dec 10;257(23):14162-7.
A new fluorescent methotrexate analogue (PT430) was synthesized as a reported ligand for dihydrofolate reductase. The analogue was prepared by attachment of lysine in place of the glutamate side chain of methotrexate and conjugation to fluorescein isothiocyanate via the epsilon-amino group of lysine. Spectrophotometric enzyme inhibition assays showed PT430 to be about one-tenth as potent as methotrexate against either Lactobacillus casei or L1210 mouse leukemia enzyme; competitive radioligand binding assays using tritiated methotrexate gave similar results. In assays of L1210 cell proliferation in culture, on the other hand, PT430 was 100-fold less toxic than methotrexate. In dilute solution, the fluorescence intensity of PT430 was 5-fold lower than that of equimolar fluorescein and diminished with decreasing pH. On complexation with dihydrofolate reductase, however, fluorescence intensity was enhanced 3- to 5-fold depending on the pH. Measurement of fluorescence increase with added ligand provided data for the determination of the stoichiometric ratio, dissociation constant, and extent of fluorescence enhancement. Specificity of PT430 for methotrexate binding sites was indicated by the observation of decreased fluorescence uptake in PT430-treated L1210 cells in the presence of methotrexate. Fluorescence uptake occurred faster, and to a greater extent, in methotrexate-resistant dihydrofolate reductase overproducing L1210/R6 cells than in the methotrexate-sensitive parent line. Therefore, PT430 may be used as a flow cytometry probe to detect methotrexate resistance based on dihydrofolate reductase overproduction.
一种新的荧光甲氨蝶呤类似物（PT430）被合成出来，作为一种已报道的二氢叶酸还原酶配体。该类似物是通过用赖氨酸取代甲氨蝶呤的谷氨酸侧链，并经由赖氨酸的ε-氨基与异硫氰酸荧光素偶联而制备的。分光光度法酶抑制试验表明，PT430对干酪乳杆菌或L1210小鼠白血病酶的效力约为甲氨蝶呤的十分之一；使用氚标记甲氨蝶呤的竞争性放射性配体结合试验给出了类似结果。另一方面，在培养的L1210细胞增殖试验中，PT430的毒性比甲氨蝶呤低100倍。在稀溶液中，PT430的荧光强度比等摩尔荧光素低5倍，且随pH值降低而减弱。然而，与二氢叶酸还原酶络合时，荧光强度根据pH值增强3至5倍。测量添加配体后的荧光增加为确定化学计量比、解离常数和荧光增强程度提供了数据。在甲氨蝶呤存在的情况下，PT430处理的L1210细胞中荧光摄取减少，这表明PT430对甲氨蝶呤结合位点具有特异性。与甲氨蝶呤敏感的亲本细胞系相比，在对甲氨蝶呤耐药的二氢叶酸还原酶过量产生的L1210/R6细胞中，荧光摄取更快且程度更大。因此，PT430可作为一种流式细胞术探针，用于基于二氢叶酸还原酶过量产生来检测甲氨蝶呤耐药性。
