Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022;12(6):479-486.
doi: 10.34172/bi.2022.23477. Epub 2022 Oct 31.

Enhanced control of bioactivity of tissue plasminogen activator (tPA) through domain-directed enzymatic oxidation of terminal galactose

Wael A Mahdi  1 , Mohammad S Absar  2 , Suna Choi  2 , Victor C Yang  3   4 , Young M Kwon  5
Affiliations

Enhanced control of bioactivity of tissue plasminogen activator (tPA) through domain-directed enzymatic oxidation of terminal galactose

Wael A Mahdi et al. Bioimpacts. 2022.

Abstract

Introduction: In targeted enzyme prodrug constructs, it is critical to control the bioactivity of the drug in its prodrug form. The preparation of such constructs often involves conjugation reactions directed to functional groups on amino acid side chains of the protein, which result in random conjugation and incomplete control of bioactivity of a prodrug, which may result in significant nontarget effect. Thus, more specific method of modification is desired. If the drug is a glycoprotein, enzymatic oxidation may offer an alternative approach for therapeutic glycoproteins. Methods: Tissue plasminogen activator (tPA), a model glycoprotein enzyme, was treated with galactose oxidase (GO) and horseradish peroxidase, followed by thiolation reaction and conjugation with low molecular weight heparin (LMWH). The LMWH-tPA conjugate was isolated by ion-exchange chromatography followed by centrifugal filtration. The conjugate was characterized for its fibrinolytic activity and for its plasminogen activation through an indirect amidolytic assay with a plasmin-specific substrate S-2251 when LMWH-tPA conjugate is complexed with protamine-albumin conjugate, followed by triggered activation in the presence of heparin. Results: LMWH-tPA conjugate prepared via enzymatic oxidation retained ~95% of its fibrinolytic activity with respect to native tPA. Upon complexation with protamine-albumin conjugate, the activity of LMWH-tPA was effectively inhibited (~90%) whereas the LMWH-tPA prepared by random thiolation exhibited ~55% inhibition. Addition of heparin fully generated the activities of both conjugates. Conclusion: The tPA was successfully modified via enzymatic oxidation by GO, resulting in enhanced control of its activity in the prodrug construct. This approach can be applied to other therapeutic glycoproteins.

Keywords: Glycoprotein oxidation; Pretargeting; Thrombolytic drug; Triggered release; tPA.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interests.

Figures

Fig. 1
Fig. 1
Fig. 2
Fig. 2
Fig. 3
Fig. 3
See this image and copyright information in PMC

References

    1. Kreitman RJ. Recombinant toxins for the treatment of cancer. CurrOpin Mol Ther. 2003;5:44–51. - PubMed
    1. Akbari B, Farajnia S, Ahdi Khosroshahi S, Safari F, Yousefi M, Dariushnejad H, et al. Immunotoxins in cancer therapy: Review and update. Int Rev Immunol. 2017;36(#):207–19. doi: 10.1080/08830185.2017.1284211. - DOI - PubMed
    1. Dean SN, Turner KB, Medintz IL, Walper SA. Targeting and delivery of therapeutic enzymes. TherDeliv. 2017;8:577–95. doi: 10.4155/tde-2017-0020. - DOI - PubMed
    1. Xiong MP, Kwon GS. PEGylation of yeast cytosine deaminase for pretargeting. J Pharm Sci. 2005;94:1249–58. doi: 10.1002/jps.20354. - DOI - PubMed
    1. Ke S, Wright JC, Kwon GS. Avidin-biotin-PEG-CPA complexes as potential EPR-directed therapeutic protein carriers: preparation and characterization. Bioconjug Chem. 2007;18:1644–50. doi: 10.1021/bc700182t. - DOI - PubMed

LinkOut - more resources