Comparisons of biophysical properties and bioactivities of mono-PEGylated endostatin and an endostatin analog

Background:Endostatin（ES） is a well-established potent endogenous antiangiogenic factor.An ES variant,called zinc-binding protein-ES（ZBP-ES）,is clinically available;however,its use is limited by rapid renal clearance and short residence time.PEGylation has been exploited to overcome these shortcomings,and mono-PEGylated ES（called M2ES） as well as mono-PEGylated ZBP-ES（MZBP-ES） are developed in our study.This study aimed to compare the biophysical properties and biological effects of M2ES and MZBP-ES to evaluate their druggability.Methods:Circular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M2ES and MZBP-ES.Their resistance to trypsin digestion and guanidinium chloride（GdmCl）-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence,respectively.The biological effects of M2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays,and the uptake of M2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence.Results:Structural analyses revealed that M2ES has a more compact tertiary structure than MZBP-ES.Moreover,M2ES was more resistant to trypsin digestion and GdmCI-induced unfolding compared with MZBP-ES.In addition,although M2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells,M2ES displayed an increased ability to enter cells compared with MZBP-ES,possibly caused by the enhanced interaction with nucleolin.Conclusions:M2ES has a more compact tertiary structure,is more stable for trypsin digestion and GdmCI-induced unfolding,exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES,indicating that M2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.