Kuefner U, Lohrmann U, Montejano Y D, Vitols K S, Huennekens F M
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037.
Biochemistry. 1989 Mar 7;28(5):2288-97. doi: 10.1021/bi00431a047.
Methotrexate (MTX) alpha-peptides containing representative neutral (alanine), acidic (aspartic acid), and basic (arginine) amino acids were synthesized by a regiospecific route. Purity and authenticity of MTX-Ala, MTX-Asp, and MTX-Arg were established by TLC, HPLC, elemental analysis, and NMR and absorbance spectra. These peptides were hydrolyzed by carboxypeptidases to yield MTX and the amino acids. Reactions were monitored by using a ninhydrin assay for the amino acids and HPLC and spectrophotometric assays for MTX. Pancreatic carboxypeptidase A (CP-A) hydrolyzed MTX-Ala and, at a much slower rate, MTX-Asp and MTX-Arg. MTX-Ala was also a substrate for pancreatic carboxypeptidase B (CP-B); marginal activity was observed with this enzyme and MTX-Arg. Human serum hydrolyzed only MTX-Arg; biphasic inhibition of this activity by 2-(mercaptomethyl)-3-(guanidinoethyl)thiopropionate was consistent with the known presence of two types of endogenous carboxypeptidase (CP-N). Cytotoxicity of the MTX peptides toward L1210 cells in culture was enhanced considerably in the presence of the appropriate carboxypeptidases. MTX-Ala was much less toxic than MTX (ID50 values of 2.0 X 10(-6) M and 2.4 x 10(-8) M, respectively), but in the presence of CP-A the ID50 of the peptide improved to 8.5 X 10(-8) M. Similar results were obtained with MTX-Asp/CP-A and MTX-Ala/CP-B combinations. MTX-Arg showed good cytotoxicity (ID50 of 5.0 X 10(-8) M), due to CP-N activity in the fetal bovine serum of the culture medium; inclusion of CP-B lowered the ID50 to that of MTX. Possible clinical uses of MTX peptides are discussed.
通过区域特异性途径合成了含有代表性中性（丙氨酸）、酸性（天冬氨酸）和碱性（精氨酸）氨基酸的甲氨蝶呤（MTX）α-肽。通过薄层层析（TLC）、高效液相色谱（HPLC）、元素分析、核磁共振（NMR）和吸收光谱确定了MTX-丙氨酸、MTX-天冬氨酸和MTX-精氨酸的纯度和真实性。这些肽被羧肽酶水解产生MTX和氨基酸。通过使用茚三酮法检测氨基酸以及HPLC和分光光度法检测MTX来监测反应。胰羧肽酶A（CP-A）水解MTX-丙氨酸，水解MTX-天冬氨酸和MTX-精氨酸的速度则慢得多。MTX-丙氨酸也是胰羧肽酶B（CP-B）的底物；观察到该酶对MTX-精氨酸有微弱活性。人血清仅水解MTX-精氨酸；2-（巯基甲基）-3-（胍基乙基）硫代丙酸对该活性的双相抑制与已知存在两种内源性羧肽酶（CP-N）一致。在适当的羧肽酶存在下，培养物中MTX肽对L1210细胞的细胞毒性显著增强。MTX-丙氨酸的毒性远低于MTX（半数抑制浓度（ID50）值分别为2.0×10⁻⁶ M和2.4×10⁻⁸ M），但在CP-A存在下，该肽的ID50提高到8.5×10⁻⁸ M。MTX-天冬氨酸/CP-A和MTX-丙氨酸/CP-B组合也得到了类似结果。由于培养基胎牛血清中的CP-N活性，MTX-精氨酸显示出良好的细胞毒性（ID50为5.0×10⁻⁸ M）；加入CP-B可使ID50降至MTX的水平。讨论了MTX肽可能的临床用途。
