Department of Applied Biology and Chemical Technology, Lo Ka Chung Research Centre for Natural Anti-Cancer Drug Development and State Key Laboratory of Chemical Biology and Drug Discovery, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
Appl Microbiol Biotechnol. 2020 May;104(9):3921-3934. doi: 10.1007/s00253-020-10484-4. Epub 2020 Mar 6.
L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion-enriched human arginase I mutant (HAI) was Co > Ni ≫ Mn. The overall k/K values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. KEY POINTS: • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. Graphical abstract.
精氨酸（L-Arg）耗竭在癌症治疗中引起了广泛关注。虽然两种精氨酸耗竭酶，即精氨酸脱亚氨酶（ADI）和人精氨酸酶 I，正在进行临床试验，但随机的 PEG 化位点、重金属作为辅助因子的低效率和免疫原性限制了这些药物的性能，并导致均相生产困难。在这里，我们筛选了十种催化金属离子，并通过将半胱氨酸残基与 PEG-马来酰亚胺缀合，成功地生产了一种定点单 PEG 化的人精氨酸酶 I 突变体，从而最小化活性降低并产生均相产物。富含金属离子的人精氨酸酶 I 突变体（HAI）的催化效率趋势为 Co>Ni≫Mn。Co-HAI 和 Ni-HAI 的总体 k/K 值分别比 Mn-HAI 高约 8.7-和 5.2 倍。此外，酶动力学和圆二色性光谱的结果表明，20 或 40 kDa 的线性和支链 PEG 附着在 HAI 表面上不会影响酶活性和蛋白质二级结构。体外研究表明，Co-HAI-PEG20L 和 Ni-HAI-PEG20L 均抑制了八种类型的癌细胞系的生长。在小鼠中的药效学研究表明，Co-HAI-PEG20L 以 13 mg/kg 和 Ni-HAI-PEG20L 以 15 mg/kg 的剂量腹腔注射一次后，能够将 L-Arg 水平维持在检测限以下超过 120 小时。注射后 5 天内，小鼠体重可恢复正常水平，表明这些剂量可耐受。因此，Ni-HAI-PEG20L 和 Co-HAI-PEG20L 都是很有前途的癌症治疗候选药物。关键点：• 成功应用于人精氨酸酶 I 突变体（HAI）的单 PEG 化。• Co 和 Ni 富集的 HAI 的催化效率高于野生型。• Co 和 Ni-HAI-PEG20L 抑制至少八种类型的癌细胞系。• Co 和 Ni-HAI-PEG20L 能够实现每周 L-Arg 的耗竭。
