Abstract
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 Cys45 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 Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM 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.
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Acknowledgments
We thank Professor Ming-Cheung Chow for the provision of DMS114 cell line. We also thank Athenex company for co-operation. Moreover, we would like to thank Dr. Yu-Wai Chen for giving comments on the manuscript. Furthermore, we thank Dr. Siu-Lun Leung for helping with the MTT assays on MDA-MB-231, A375, and 4T1 cancer cell lines.
Funding
This work was supported by the Hong Kong Research Grants Council (RGC) General Research Fund (PolyU 5017/13P), University Supporting Fund (1-BBAE), Project of Strategic Importance (1-ZE18 & 1-ZE21), the Lo Ka Chung Charitable Foundation Limited (847E), and PolyU Strategic Development Special Project (1-ZVH9).
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KW, YL, and WL conceived and designed the research. SC, CK, ST, MC, and PS conducted the experiments. SC, YL, and WL analyzed data and wrote the manuscript. All authors read and approved the manuscript.
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All animal experiments were conducted with licenses issued by the Department of Health of the Hong Kong Government as stipulated by the Animals (control of experiments) ordinance (Cap. 340), under the approval no. 12/23 granted by the animal ethic sub-committee of the Polytechnic University of Hong Kong and following the institutional guidelines for care and use of animals. The normal BALB/c mice were obtained from the Central Animal Facilities (CAF) of the Polytechnic University of Hong Kong.
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Chung, SF., Kim, CF., Tam, SY. et al. A bioengineered arginine-depleting enzyme as a long-lasting therapeutic agent against cancer. Appl Microbiol Biotechnol 104, 3921–3934 (2020). https://doi.org/10.1007/s00253-020-10484-4
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DOI: https://doi.org/10.1007/s00253-020-10484-4