Abstract
Honey is a natural product produced by honeybees, which has been used not only as food but also as a medicine by humans for thousands of years. In this study, 60 kDa protein was purified from Pakistani Sidr honey named as SHP-60 (Sidr Honey Protein-60), and its antioxidant potential and the effect of Bevacizumab with purified protein on in vitro angiogenesis using human umbilical vein endothelial cells (HUVEC) were investigated. We further validated the molecular protein-protein (SHP-60 with Bevacizumab) interactions through in silico analysis. It showed very promising antioxidant activity by reducing 2,2-diphenyl-1-picrylhydrazyl free radicals with a maximum of 83% inhibition at 50 μM and an IC50 of 26.45 μM statistically significant (**p < 0.01). Angiogenesis is considered a hallmark of cancer, and without it, the tumor cannot grow or metastasize. Bevacizumab, SHP-60, and both in combination were used to treat HUVEC, and the MTT assay was used to assess cell viability. To demonstrate in vitro angiogenesis, HUVEC was grown on Geltrex, and the formation of endotubes was examined using a tube formation assay. HUVEC viability was dose-dependently decreased by Bevacizumab, SHP-60, and both together. Bevacizumab and SHP-60 both inhibited angiogenesis in vitro, and their combination displayed levels of inhibition even higher than those of Bevacizumab alone. We investigated the protein-protein molecular docking interactions and molecular dynamics simulation analysis of MRJP3 (major royal jelly protein 3) similar to SHP-60 in molecular weight with both the heavy chain (HC) and light chain (LC) of Bevacizumab. We found significant interactions between the LC and MRJP3, indicating that ASN468, GLN470, and ASN473 of MRJP3 interact with SER156, SER159, and GLU161 of LC of Bevacizumab. The integration of experimental data and computational techniques is believed to improve the reliability of the findings and aid in future drug design.