VEGFR2 Mimicking Peptide Inhibits the Proliferation of Human Umbilical Vein Endothelial Cells (Huvecs) by Blocking VEGF


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Abstract

Introduction:A variety of key human physiological processes rely on angiogenesis, ranging from reproduction and fetal growth to wound healing and tissue repair. Furthermore, this process significantly contributes to tumor progression, invasion, and metastasis. As the strongest inducer of angiogenesis, Vascular Endothelial Growth Factor (VEGF) and its receptor (VEGFR) are targets of therapeutic research for blocking pathological angiogenesis.

Objective: Preventing the interaction between VEGF and VEGFR2 by a peptide is a promising strategy for developing antiangiogenic drug candidates. This study was aimed at designing and evaluating VEGF-targeting peptides using in silico and in vitro techniques.

Methods: The VEGF binding site of VEGFR2 was considered a basis for peptide design. The interaction of VEGF and all three peptides derived from VEGFR2 were analyzed using ClusPro tools. In a complex with VEGF, the peptide with a higher docking score was evaluated to confirm its stability using molecular dynamics (MD) simulation. The gene coding for the selected peptide was cloned and expressed in E. coli BL21. The bacterial cells were cultured on a large scale, and the expressed recombinant peptide was purified using Ni-NTA chromatography. Refolding of the denatured peptide was carried out by the stepwise removal of the denaturant. The reactivity of peptides was confirmed using western blotting and enzyme-linked immunosorbent assay (ELISA) assays. Finally, the inhibition potency of the peptide on human umbilical vein endothelial cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay

Results: Among three peptides, the peptide with the best docking pose and the highest affinity for VEGF was selected for further studies. Then the stability of the peptide was confirmed over the 100 ns MD simulation. After in silico analyses, the selected peptide was presented for in vitro analysis. Expression of the selected peptide in E. coli BL21 resulted in a pure peptide with a yield of approximately 200 µg/ml. Analysis by ELISA revealed the high reactivity of the peptide with VEGF. Western blot analysis confirmed the specific reactivity of selected peptides with VEGF. The MTT assay revealed the growth inhibitory effect of the peptide on human umbilical vein endothelial cells with an IC50 value of 247.8 µM.

Conclusion: In summary, the selected peptide demonstrated a promising inhibitory effect on human umbilical vein endothelial cells that could be a valuable anti-angiogenic candidate for further assessment. Additionally, these in silico and in vitro data provide new insights into peptide design and engineering.

About the authors

Samaneh Ghasemali

Department of Medical Biotechnology, School of Advanced Medical Sciences,, Tabriz University of Medical Sciences

Email: info@benthamscience.net

Abolfazl Barzegar

Department of Biology, Faculty of Natural Science, University of Tabriz

Email: info@benthamscience.net

Safar Farajnia

Drug Applied Research Center, Tabriz University of Medical Sciences

Author for correspondence.
Email: info@benthamscience.net

Mohammad Rahmati

Department of Clinical Biochemistry, Faculty of Medicine,, Tabriz University of Medical Sciences

Email: info@benthamscience.net

Babak Negahdari

Medical Biotechnology Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS)

Email: info@benthamscience.net

Ali Etemadi

Medical Biotechnology Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS)

Email: info@benthamscience.net

Atefeh Nazari

Biotechnology Research Center, Tabriz University of Medical Sciences,

Email: info@benthamscience.net

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