Chemosynthesis and characterization of site-specific N-terminally PEGylated Alpha-momorcharin as apotential agent

Abstract

Alpha-momorcharin (α-MC), a type I ribosome-inactivating protein (RIP) isolated from Momordica charantia seeds, has been extensively studied for its antitumor, antiviral and antifungal activities. However, as an exogenous protein, problems associated with short half-life and strong immunogenicity have limited its clinical application. Poly (ethylene glycol) (PEG), as a polyether compound, is a well established and efficient modifier to develop it as a potential agent. Nevertheless, conventional PEGylation is not site-controlled and the conjugates are often not homogenous due to the generation of multi-PEGylated derivatives. To obtain a homogenous mono-PEGylated α-MC, the PEGylation was carried out by coupling a 20 kDa mPEG-butyraldehyde (mPEG-ALD) with α-MC. The product was separated and purified by MacroCap SP chromatography. Results from SDS-PAGE and MALDI-TOF MS revealed that the PEGylated α-MC consisted of one molecule mPEG and α-MC. Edman degradation confirmed that the N-terminal residue of α-MC was successfully coupled with mPEG-ALD. The mono-PEGylated α-MC possessed an extremely similar secondary structure to native α-MC through spectral analyses. In addition, it also showed low immunogenicity by double immunodiffusion and preserved moderate antitumor activity to three kinds of tumor cell lines in vitro. Finally, trypsin resistance was also considerably improved.

Isomorphic Fluorescent Nucleosides Facilitate Real‐Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins

“…While lethal to mammalian cells, RIPs can also display antiviral features [8] and can inhibit animal and plant viruses such as HIV‐1, [9] poliovirus [10] and tobacco etch virus [11] . RIP‐conjugated toxins have also been developed as potential antitumor agents [12,13] . Their unique and surgically directed cellular activity, as well as potency and potential utility as biowarfare agents, have therefore attracted considerable interest over the years, [14] aiming particularly at the development of effective strategies for real time activity monitoring and detection [15] …”

“…[11] RIP-conjugated toxins have also been developed as potential antitumor agents. [12,13] Their unique and surgically directed cellular activity, as well as potency and potential utility as biowarfare agents, have therefore attracted considerable interest over the years, [14] aiming particularly at the development of effective strategies for real time activity monitoring and detection. [15] Prior studies have utilized enzyme-linked immunosorbent assay (ELISA), [16] immuno-PCR [17] and mass spectrometry to assess RIP activity.…”

Biological Activities of Ribosome-Inactivating Proteins

“…Mono-, di-, tri-PEGlyated α-MMC and MAP30 dramatically reduced the immunogenicity and maintained biological activities [112,113]. The enzymatic activity assay showed that mono-PEGylated α-MMC strongly inhibited the growth of human cervix adenocarcinoma cells [116]. Sun et al provided a method to isolate high purity mono-mPEGylated MAP30 and α-MMC, which can be useful for further RIP drug examination [111].…”

Engineering of Ribosome-inactivating Proteins for Improving Pharmacological Properties

“…Mono-, di-, tri-PEGlyated α-MMC and MAP30 dramatically reduced the immunogenicity and maintained biological activities [112,113]. The enzymatic activity assay showed that mono-PEGylated α-MMC strongly inhibited the growth of human cervix adenocarcinoma cells [116]. Sun et al provided a method to isolate high purity mono-mPEGylated MAP30 and α-MMC, which can be useful for further RIP drug examination [111].…”

“…The plasma half-life of α-MMC was sharply increased from 6.2-7.5 h to 52-87 h [114]. The homogeneity of PEGlyated α-MMC was further improved by site-specific conjugation of mPEG-ALD [116]. Due to steric hindrance of active sites, PEGylation may lead to biological activity reduction, which can be compensated by longer plasma half-life [119].…”