Kuefner U, Lohrmann U, Montejano Y, Vitols K S, Huennekens F M
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037.
Adv Enzyme Regul. 1988;27:3-13. doi: 10.1016/0065-2571(88)90005-2.
MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.
已制备了氨基酸与α - 羧基相连的甲氨蝶呤（MTX）肽，并在用蛋白水解酶处理前后检测了其细胞毒性。按照Rosowsky和Montgomery的一般方法，通过区域特异性途径合成了丙氨酸、天冬氨酸和精氨酸衍生物（MTX - ala、MTX - asp和MTX - arg）。每种化合物的产率都很高，其纯度通过薄层色谱法（TLC）、高效液相色谱法（HPLC）、吸收光谱和元素分析得以确定。MTX肽不会被多种蛋白水解酶（如胰蛋白酶、纤溶酶、尿激酶、氨肽酶）水解。然而，胰羧肽酶A能轻易水解MTX - ala，缓慢水解MTX - asp，而完全不水解MTX - arg。MTX - ala以及在较小程度上MTX - arg是胰羧肽酶B的作用底物。MTX - arg也会被人血清中的内源性羧肽酶N水解。在使用四氮唑染料的微量培养测定系统中，测定了这些MTX肽对L1210细胞的细胞毒性。与MTX（ID50 = 2.4×10⁻⁸M）相比，MTX - ala的细胞毒性较弱（ID50 = 2.0×10⁻⁶M）。当在羧肽酶A存在的情况下测试MTX - ala时，ID50值提高到了8.5×10⁻⁸M。MTX - arg的ID50为5.0×10⁻⁸M，鉴于其易被培养基中胎牛血清存在的羧肽酶活性水解，这并不意外。加入羧肽酶B后，ID50值降至2.5×10⁻⁸M。讨论了MTX肽可能的临床用途。
