Jones D A, Cummings J, Langdon S P, MacLellan A, Smyth J F
Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital, Edinburgh, U.K.
Biochem Pharmacol. 1995 Aug 25;50(5):585-90. doi: 10.1016/0006-2952(95)00191-2.
H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2, a broad spectrum neuropeptide growth factor antagonist (antagonist G), is soon to enter a phase I clinical trial for the treatment of small-cell lung cancer (SCLC). The pre-clinical pharmacology of this peptide has revealed that its metabolism proceeds from the C-terminus via deamidation. In this study the class of enzyme responsible for the degradation of antagonist G has been characterized. Tissue distribution studies on the enzyme have shown it to be very widespread with high specific activity being detected in the spleen, kidney, H69 SCLC xenograft and liver (12.64, 9.58, 8.00 and 6.94 nmols G/mg protein/hr, respectively). HPLC gel filtration indicated that the G-deamidase enzyme had an apparent molecular mass of 81 kDa. The sub-cellular distribution of the enzyme using differential centrifugation indicates that it is largely soluble with > 85% of the activity located in the cytosolic fraction. The distribution of activity towards antagonist G closely resembles that of esterase and acid carboxypeptidase activity, two activities, along with deamidase activity, known to be possessed by serine carboxypeptidases. Studies using a range of protease inhibitors showed clear inhibition of metabolism by phenylmethylsulphonylfluoride and benzyloxycarbonylphenylalanine chloromethylketone, indicating that the enzyme is a chymotrypsin-like serine carboxypeptidase. This knowledge of the enzyme will be invaluable in the further development of antagonist G and similar compounds. Moreover, the widespread distribution of this enzyme together with its broad specificity for C-terminal group suggests that it should be given serious consideration when designing C-terminally modified peptide drugs.
H-精氨酸-D-色氨酸-N-甲基苯丙氨酸-D-色氨酸-亮氨酸-甲硫氨酸-NH2，一种广谱神经肽生长因子拮抗剂（拮抗剂G），即将进入治疗小细胞肺癌（SCLC）的I期临床试验。该肽的临床前药理学研究表明，其代谢从C端通过脱酰胺作用进行。在本研究中，已对负责降解拮抗剂G的酶类进行了表征。对该酶的组织分布研究表明，它分布非常广泛，在脾脏、肾脏、H69 SCLC异种移植瘤和肝脏中检测到高比活性（分别为12.64、9.58、8.00和6.94 nmol G/ mg蛋白/小时）。HPLC凝胶过滤表明，G-脱酰胺酶的表观分子量为81 kDa。使用差速离心法对该酶进行亚细胞分布研究表明，它主要是可溶的，超过85%的活性位于胞质部分。对拮抗剂G的活性分布与酯酶和酸性羧肽酶活性非常相似，已知丝氨酸羧肽酶具有这两种活性以及脱酰胺酶活性。使用一系列蛋白酶抑制剂的研究表明，苯甲基磺酰氟和苄氧羰基苯丙氨酸氯甲基酮可明显抑制代谢，表明该酶是一种胰凝乳蛋白酶样丝氨酸羧肽酶。对该酶的了解在拮抗剂G和类似化合物的进一步开发中将具有重要价值。此外，该酶的广泛分布及其对C端基团的广泛特异性表明，在设计C端修饰的肽类药物时应予以认真考虑。
