Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol

Yao Meng,^1,2 Shuangfeng Liu,¹ Juan Li,³ Yanfa Meng,³ Xiaojun Zhao<sup>2,4

1</sup>School of Medical Laboratory Science, Chengdu Medical College, Chengdu, China; ²West China Hospital Laboratory of Nanomedicine and Institute for Nanobiomedical Technology and Membrane Biology, Sichuan University, Chengdu, China; ³Key Laboratory of Bio-resources and Eco-environment Ministry of Education/Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu, China; ⁴Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

Background: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)₂-Lys-NHS (20 kDa).
Methodology/principal findings: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts.
Conclusion/significance: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.

Keywords: ribosome-inactivating proteins, alpha-momorcharin, momordica anti-HIV protein, antitumor, antivirus, (mPEG)₂-Lys-NHS (20 kDa), immunogenicity