==== Front PLoS One PLoS One plos PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0287577 PONE-D-23-06800 Research Article Biology and Life Sciences Biochemistry Biochemical Simulations Biology and Life Sciences Computational Biology Biochemical Simulations Biology and Life Sciences Physiology Cardiovascular Physiology Angiogenesis Biology and Life Sciences Developmental Biology Angiogenesis Biology and Life Sciences Toxicology Toxicity Medicine and Health Sciences Pathology and Laboratory Medicine Toxicology Toxicity Physical Sciences Chemistry Computational Chemistry Molecular Dynamics Biology and Life Sciences Toxicology Predictive Toxicology Medicine and Health Sciences Pathology and Laboratory Medicine Toxicology Predictive Toxicology Physical Sciences Chemistry Computational Chemistry Molecular Docking Computer and Information Sciences Software Engineering Computer Software Engineering and Technology Software Engineering Computer Software Biology and Life Sciences Biophysics Biophysical Simulations Physical Sciences Physics Biophysics Biophysical Simulations Biology and Life Sciences Computational Biology Biophysical Simulations Computer simulation approach to the identification of visfatin-derived angiogenic peptides Visfatin-derived angiogenic peptides by computer simulation Choi Ji Myung Conceptualization Writing – original draft 1 2 https://orcid.org/0000-0001-8716-5235 Vuppala Srimai Conceptualization Methodology Writing – original draft 3 Park Min Jung Conceptualization Investigation Methodology Writing – original draft 1 4 Kim Jaeyoung Data curation Methodology 3 Jegal Myeong-Eun Data curation Methodology 5 Han Yu-Seon Methodology 5 Kim Yung-Jin Data curation Methodology 5 6 Jang Joonkyung Software Supervision Writing – review & editing 3 ‡ * Jeong Min-Ho Supervision Writing – review & editing 2 ‡ * https://orcid.org/0000-0002-8214-3472 Joo Bo Sun Conceptualization Supervision Writing – review & editing 1 4 ‡ * 1 Lab-to-Medi CRO Inc., Seoul, Republic of Korea 2 Department of Microbiology, Dong-A University College of Medicine, Busan, Republic of Korea 3 Department of Nanoenergy Engineering, Pusan National University, Busan, Republic of Korea 4 The Korea Institute for Public Sperm Bank, Busan, Republic of Korea 5 Korea Nanobiotechnology Center, Pusan National University, Busan, Republic of Korea 6 Department of Molecular Biology, Pusan National University, Busan, Republic of Korea Sever Belgin Editor Anadolu University: Anadolu Universitesi, TURKEY Competing Interests: The authors have declared that no competing interests exist. ‡ JJ, MHJ and BSJ also contributed equally to this work. * E-mail: jkjang@pusan.ac.kr (JJ); mhjeong@dau.ac.kr (MHJ); bosunoo@hanmail.net (BSJ) 29 6 2023 2023 18 6 e02875777 3 2023 7 6 2023 © 2023 Choi et al 2023 Choi et al https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Angiogenesis plays an essential role in various normal physiological processes, such as embryogenesis, tissue repair, and skin regeneration. Visfatin is a 52 kDa adipokine secreted by various tissues including adipocytes. It stimulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis. However, there are several issues in developing full-length visfatin as a therapeutic drug due to its high molecular weight. Therefore, the purpose of this study was to develop peptides, based on the active site of visfatin, with similar or superior angiogenic activity using computer simulation techniques.Initially, the active site domain (residues 181∼390) of visfatin was first truncated into small peptides using the overlapping technique. Subsequently, the 114 truncated small peptides were then subjected to molecular docking analysis using two docking programs (HADDOCK and GalaxyPepDock) to generate small peptides with the highest affinity for visfatin. Furthermore, molecular dynamics simulations (MD) were conducted to investigate the stability of the protein-ligand complexes by computing root mean square deviation (RSMD) and root mean square fluctuation(RMSF) plots for the visfatin-peptide complexes. Finally, peptides with the highest affinity were examined for angiogenic activities, such as cell migration, invasion, and tubule formation in human umbilical vein endothelial cells (HUVECs). Through the docking analysis of the 114 truncated peptides, we screened nine peptides with a high affinity for visfatin. Of these, we discovered two peptides (peptide-1: LEYKLHDFGY and peptide-2: EYKLHDFGYRGV) with the highest affinity for visfatin. In an in vitrostudy, these two peptides showed superior angiogenic activity compared to visfatin itself and stimulated mRNA expressions of visfatin and VEGF-A. These results show that the peptides generated by the protein-peptide docking simulation have a more efficient angiogenic activity than the original visfatin. National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) No. 2020R1A2C2014089 https://orcid.org/0000-0002-8214-3472 Joo Bo Sun This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A2C2014089). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files. Data Availability All relevant data are within the manuscript and its Supporting Information files. ==== Body pmcIntroduction The fundamental processes by which new blood vessels are formed in the body can be classified into vasculogenesis and angiogenesis. Vasculogenesis refers to the de novo formation of blood vessels from endothelial precursor cells in the body, and angiogenesis refers to the formation of new blood vessels from pre-existing vasculature. Angiogenesis is known to play an essential role in various normal physiological processes, such as embryogenesis, tissue repair, and organ regeneration. Angiogenesis also occurs in tumor cells to supplyblood for tumor growth and metastasis [1]. In addition, angiogenesis plays a very important role in skin regeneration, including wound healing and tissue recovery [2, 3], and in physiological events such as follicle development and embryo implantation [4–6]. Ovarian aging is a natural and physiological aging process characterized by a rapid decline in the oocyte quality along with a decrease in the number of follicles. It is a representative clinically unmet needfor infertility treatment. However, many recent studies have reported that ovarian aging can be reversed if an appropriate microenvironment is present in the ovary [7, 8]. One of the suggested strategies for overcoming ovarian aging is the activation of ovarian angiogenesis [9–12]. Our earlier studies have showed that the administration of visfatin during superovulation restores oocyte developmental competency and fertility potential in aged female mice [13, 14]. Visfatin was originally known as a pre-B cell colony-enhancing factor that promotes the growth of B-lymphocytes precursor cells [15]. However, it has recently been identified as an adipokine secreted from various cells such as macrophages, amniotic epithelial cells, human granulosa cells, and adipocytes [16–18]. In addition, since the first report of Kim et al. on the angiogenesis-promoting function of visfatin [19], many studies have shown that visfatin not only induces the production of the vascular endothelial growth factor (VEGF), a representative angiogenic factor, but also stimulates angiogenesis, including proliferation, migration, and metastasis of vascular endothelial cells [20–22]. However, high molecular weight of visfatin is a major limitation to its development as a therapeutic drug because full-length proteins have several inherent disadvantages, such as immunogenicity, lower stability, and loss of bioactivity [23]. In contrast, therapeutic peptides consisting of short-length amino acid sequence (3 to 20) have exhibited beneficial effects for the treatment of several health conditions [24, 25]. The peptides offer specific advantages such as good efficacy, good safety, low immunogenicity, high membrane permeability, and low cost compared to therapeutic proteins and antibodies [25–27]. For this reason, interest in the field of therapeutic peptides, including marine peptides, has increased in recent years. To date, more than 170 peptides are in active clinical development with many more in the preclinical stages [28–30]. In particular, recent improvements in peptide screening and computational biology have increased the demand for peptide drug discovery [25]. Most natural peptides act as agonists on their receptors, where the interaction between the peptide and the receptor is the first step to initiate an active response within the cell [31]. The protein-peptide docking can be simulated with computer-aided drug design (CADD) technologyand is very useful in deriving small agonist peptide sequences that can produce biological efficaciessimilar to that of natural ligands [32]. Therefore, the purpose of this study was to develop peptides,based on the active site of visfatin, with similar or superior angiogenic activity using computer simulation techniques. Materials and methods Construction of the peptide library The following systematic approach was used to determine significant hotspots and active spots in the visfatin sequences. First, a three-dimensional structural analysis of visfatin showed that it was composed of two structural domains, and the active site was located in the second structural domain (from amino acid residues 181 to 390), which was defined as the active site domain (hereafter A-domain peptide) in this study [33, 34]. The A-domain peptide was then truncated into small peptides using the overlapping technique. Second, the three-dimensional structures of all the truncated peptide sequences were generated and predicted using the peptide tertiary structure prediction server with natural, non-natural,and modified residues (PEPstrMOD) [35, 36]. Finally, the designed 114 peptide sequences were subjected to molecular docking analysis to identify the hotspots in the globular A-domain peptide. Molecular docking simulations A well-validated “structure-based molecular docking” computational technique that took into consideration the three-dimensional structure of visfatin was applied to design a small natural agonist peptide sequence. The three-dimensional structure of visfatin (PDB ID: 2G95) was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB)(http://www.rcsb.org), and the amino acids of the known active site of visfatin, namely, TYR18, PHE193, TYR195, ARG196, GLY197, ASP219, HIS247, ARG311, ARG313, GLY353, and ASP354, were identifed [33].The peptides with the highest affinity for visfatin were identified using two protein-peptide docking simulation programs, High Ambiguity Driven protein-protein DOCKing (HADDOCK) [37] and Galaxyprotein–peptide docking (GalaxyPepDock) [32]. Molecular dynamics simulation of protein-ligand complexes Molecular dynamics (MD) simulations were performed on the visfatin-peptide complexes to assess their structural stability. The Amber ff19SB force field [38] and optimal point charges (OPC) water model [39] were employed to model the protein and water molecules. Each system was neutralized in a 0.15 M NaCl solution by adding Na+ and Cl- ions. All the MD simulations were carried out in an NVT ensemble (fixed number of atoms, N, a fixed volume, V, and a fixed temperature, T) with a temperature fixed at 310 K. The Nose-Hoover thermostat method was used to keep the temperatureconstant. A leap-frog algorithm with a time step of 2 fs was used to propagate the MD trajectory. We run 50 ns of data production simulations without any restraints after 500 ps of equilibration simulation. The simulation progress was saved step by step every 100 ps. The simulation systems were constructed using Chemistry at Harvard Macromolecular Mechanics-Graphical User Interface (CHARMM-GUI) [40], and all the MD simulations were implemented using the Gromacs2021.5 software [41]. In silico toxicity, angiogenic, antihypertensive profiles, and half-life predictions of peptides The toxicity profiles (hemotoxicity, cytotoxicity, and immunotoxicity) of the designed peptides were predicted using the in silico tools ToxinPred [42] and HemoPI [43]. Machine learning using support vector machine and quantitative matrix (QM) models was used to predict the toxicity of the peptides. The half-life of a peptide is related to its stability and toxicity. The PlifePred [44] and HLP [45] web servers were used to predict the half-lives of peptides in blood and intestine-like environments, respectively. The AHTPin [46] and AntiAngiopred [47] web servers were used to predict the antiangiogenic and hypertensive properties of the designed peptides. Preparation of visfatin and visfatin-derived peptides for cytotoxicity and angiogenesis assessment in vitro The two synthetic peptides (peptide-1 and peptide-2) derived from the docking simulation having the highest affinity were obtained from GL Biochem Ltd. (Shanghai, China) and used for cytotoxicity and angiogenesis assessment in vitro. The synthetic peptides were supplied as dry powders with a purity of 95% or more guaranteed by mass spectrometry and high-performance liquid chromatography (HPLC), reconstituted in dimethyl sulfoxide (DMSO) to a concentration of 1 mg/mL, and stored at -20°C until use. Commercial recombinant mouse visfatin were purchased from Adipogen Life Science, Inc. (San Diego, USA). For preparing the stock solution, visfatin was dissolved in DMSO to a concentration of 1 mg/mL, and recombinant human basic fibroblast growth factor (b-FGF) was dissolved in phosphate-buffered saline (PBS) to a concentration of50 μg/ml. These stock solutions were stored at -20°C until use. Prior to cytotoxicity and angiogenesis assay experiments, b-FGF, visfain, and visfatin-derived peptides were diluted with endothelial cell growth medium (EGM) supplemented with an EGM-Supplement Mix® (PromoCell) to achieve the working concentration. b-FGF and visfain were used as a positive control, and the EGM was used as a control. The EGM-Supplement Mix® consisted of 2% fetal bovine serum (FBS), 0.004 ml/ml endothelial cell growth Supplement, 0.1 ng/ml recombinant human epidermal growth factor (rhEGF), 1 ng/ml recombinant human b-FGF, 90 μg/ml heparin, and 1 μg/ml hydrocortisone. Culture and maintenance of human vascular endothelial cells (HUVECs) Human vascular endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany) were cultured inendothelial cell growth medium 2 (EGM-2, PromoCell) supplemented with EGM-2 Supplement Mix®(PromoCell) and 2% fetal bovine serum (FBS) for 24 hours at 37°C and 5% CO2. The EGM-2 Supplement Mix® consisted of 5 ng/ml rhEGF, 10 ng/ml recombinant human b-FGF, 20 ng/ml recombinant human insulin-like growth factor (Long R3rhIGF,), 0.5vng/ml recombinant human vascular endothelial growth factor 165 (VEGF), 1 μg/ml ascorbic acid, 22.5 μg/ml heparin, 0.2 μg/ml hydrocortisone. Cells between passages 2 and 7 were used in all experiments. MTT cell cytotoxicity assay HUVECs were grown in EGM-2 supplemented with an EGM-2 Supplement Mix® (PromoCell) containing 2% FBS at a density of 1×104 cells in 96-well plates (SPL Inc., Gyeonggido, Korea). After 24 hours, the cells were treated with 1000ng/ml visfatin (Adipogen Life Science) and various concentrations of visfatin-derived peptides (0.1, 0.5, 1.0, and 2.0 μM), and then incubated overnights in an atmosphere of 5% CO2. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay reagents (Sigma-Aldrich, St. Louis, MO, USA) were as added directly to each well to a final concentration of 0.5 mg/ml. After 4 hours, the medium was removed, the formazan crystals formed in the cells were dissolved in DMSO, and the absorbance of the formazan solution was measured using a Synergy HTX Multi-Mode Reader (BIO-TEK, Vermont, USA) with a 540 nm filter. Each sample was assayed in triplicate. In vitro trans-well invasion assay The invasion capacity of the cells was determined using a 24-well trans-well system. The upper side of the trans-well membrane was coated with 1 mg/mL Matrigel at 10 μL/well at room temperature for 1 hour. The EGM-2 medium (0.5 ml)was added to the lower part of trans-well chamber. HUVECs (2×104 cells) and EGM medium (0.1 ml) were placed on the upper compartment of the trans-well. And then b-FGF (25ng/ml), visfatin (1000 ng/ml), and visfatin-derived peptides (0.5μM and 2.0μM) were added to the upper compartment of the trans-well. Cells were incubated at 37°C and 5% CO2 for 24 hours, fixed with methanol, and stained with hematoxylin and eosin. The cells on the upper surface of the membrane were removed by wiping with a cotton swab. Cell invasion was determined by counting the number of whole cells in a single filter using optical microscopy at 40× magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times. In vitro wound healing migration assay HUVECs were seeded in 24-well plates (SPL) at a density of 2×105 cells and incubated in EGM medium supplemented with EGM-Supplement Mix® containing 1% FBS overnight. Cells were scratched using a P200 pipette tip to induce a wound. Subsequently, then b-FGF (25ng/ml), visfatin (1000 ng/ml), and visfatin-derived peptides (0.5μM and 2.0μM) were added and the culture was maintained for 18 hours to allow the cells to migrate. The migration patterns were observed using a phase-contrast microscope and images were captured. The wound diameters were photographed at 16–24 hours. Wound closure was determined using optical microscopy at 40× magnification. Migration was quantified by counting the number of cells that had moved beyond the reference line.   In vitro tube formation assays HUVECs (2×104 cells/well) were seeded on a layer of polymerized Matrigel and treated with or without visfatin-derived peptides. The Matrigel culture was incubated at 37°C in the EGM supplemented with EGM-Supplement Mix® containing 1% FBS. After 4 hours, changes in the cell morphology were observed using a phase-contrast microscope and photographed at 40× magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times. Angiogenesis was analyzed with ImageJ software using an angiogenesis analyzer (Bethesda, MD, USA). Real-time quantitative PCR (RT-qPCR) After treating HUVECs with visfatin-derived peptides for 24 hours, the total RNA was isolated from the cultured HUVECs using the Tri-RNA reagent (Favorgen Biotech Corp., Kaohsiung, Taiwan) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The mRNA expression levels of VEGF and visfatin were carried out using Applied Biosystems 7500 software (Applied Biosystems Inc., Waltham, MA, USA). Real-time PCR was performed using SYBR Green Q-PCR Master Mix (Samjung Bio Science, Daejeon, South Korea). The reaction conditions were as follows: initial denaturation at 95°C for 5 min, amplification at 95°C for 15 sec, and at 60°C for 1 min. All PCR amplifications were performed for 40 cycles. The specific primers used were as follows: 5’-AAGGGTCATCATCTCTGCCC-3’ (forward) and 5’-GTG ATGGCATGGACTGTGGT-3’ (reverse) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5’-GGAGG GTGACGGGGTGAAGG-3’ (forward) and 5’-GTCGGTGGCCAGGAGGATGTT-3’ (reverse) for visfatin; and 5’-CTACCTCCACCATGCCAAGT-3’ (forward) and 5’-AGCTGCGCTGATAGACATCC-3’ (reverse) for VEGF-A. The relative changes in the gene expression were determined using the 2-ΔΔCt method and the results were normalized to the expression of GAPDH. Statistical analysis All data are presented as mean ± standard deviation (SD). The Prism 9 program (GraphPad Inc., USA) was used for statistical analysis. The experimental results were statistically processed using the t-test and two-way ANOVA. Statistical significance was set at P value < 0.05. Results Construction of peptide library using the overlapping technique To find a hotspot or active spot in the visfatin sequence, the A-domain (residues 181–390) of visfatin was truncated into small peptidesutilizing the overlapping technique. As a result, 114 truncated small peptides were generated and categorized into three datasets based on sequence length 6 (dataset-1, 38 peptides), 7 (dataset-2, 42 peptides), and 9 (dataset-3, 34 peptides) with overlapping amino acids 3, 2, and 3, respectively (Fig 1). 10.1371/journal.pone.0287577.g001 Fig 1 Fasta sequence of active site domain (A-domain) of visfatin (491AA). Shown in blue and red is the sequence belonging to the A-domain of visfatin, respectively. Six hotspots are shown in red. Molecular docking simulationsof small peptides Two protein-peptide docking simulation programs, viz.HADDOCK and GalaxyPepDock were used to perform the molecular docking simulations of the 114 truncated peptides. The HADDOCK score is a measure of how well the peptide interacts with the active site and represents its binding capability. On the other hand, GalaxyPepDock performs similarity-based docking by finding templates from the database of experimentally determined structures and building models. The GalaxyPepDock score is thus used to evaluate similarity of interactions in newly designed protein-peptide complexes to those observed in established protein-peptide complexes found in a database, thus providing insights into their compatibility and potential binding characteristics. From the docking analysis of the 114 peptides, 27 peptide sequences showed high scores with a sequence length of 9 (dataset-3). Therefore, we considered a peptide sequence length of 9 as the standard length for further screening. From the 27 peptide sequences, we reconstructed and identified six hotspots “LEYKLHDFGYRGVSSQ, GIALIKKYYGTKDPV, IYNACEKIWGEDLRH, HSTITAWGKDHEKDAF, KFPVSENSKGYKLLPPY, and GMKQKKWSIENVSFG” in the A-domain peptide of visfatin (Fig 1) and they were considered for further analysis to identify the best agonistic peptides. Seventy-six peptide sequences with different lengths from the six hotspots were designed and subjected to docking simulation. To understand the interactions of our designed peptides, we performed the molecular docking simulations of nicotinamide mononucleotide (NMN) which is a natural ligand (agonist) of visfatin(Fig 2A). The active site residues, GLY384, with an H-bond length (H-BL) of 2.52 Å, ARG196 (H-BLs 2.63 Å and 2.56 Å), and ARG311 (H-BL 2.63 Å) formed H-bond interactions with NMN, signifying their importance in the catalytic activity. The catalytic site interactions of nine peptides among those screened (Table 1) were similar to those of natural NMN ligand. This result indicates that the nine peptides may act as potential agonists of visfatin. Of the nine peptides, peptide-1 (LEYKLHDFGY, 10AA) and peptide-2 (EYKLHDFGYRGV, 12AA) had the highest scores in the two docking programs. Fig 2B and 2C shows the molecular interactions of the two peptides within the active site of visfatin. Further, the stability of the protein-peptide complexes was confirmed from the MD simulations under solvent conditions. 10.1371/journal.pone.0287577.g002 Fig 2 Diagram of the binding pose of (A). NMN, (B). Peptide-1 (LEYKLHDFGY), and (C). Peptide-2 (EYKLHDFGYRGV). The NMN, peptide-1 and peptide-2 are drawn as sticks, and the visfatin active site residues are represented by balls and sticks. The blue-colored broken lines represent H-bond interactions. 10.1371/journal.pone.0287577.t001 Table 1 Molecular docking results of the peptide sequences similar to NMN against visfatin. Amino acid sequence HADDOCK Score GalaxyPepDock score Peptide-1 LEYKLHDFGY(10AA) -106.0 +/- 2.0 16.0 Peptide-2 EYKLHDFGYRGV(12AA) -105.9 +/- 3.5 17.0 Peptide-3 ALIKKYYGTKDPV (13AA) -111.2 +/- 2.9 10.0 Peptide-4 STITAWGKDHEKDAF (15AA) -124.3 +/- 6.5 1.0 Peptide-5 ACEKIWGEDLRH (12AA) -85.5 +/- 5.5 0.0 Peptide-6 CEKIWGEDLRH (11AA) -93.6 +/- 4.2 0.0 Peptide-7 EKIWGEDLRH (10AA) -71.6 +/- 2.0 0.0 Peptide-8 GMKQKKWSIENVSF (14AA) -97.8+/- 4.3 15.0 Peptide-9 ENSKGYKLLPPY (12AA) -82.7 +/- 2.9 18.0 Molecular dynamic simulations of the visfatin-derived peptides The molecular dynamic (MD) simulations were performed for peptide-1 and peptide-2 using Gromacs2021.5 software. The highest-scoring conformations of the protein-ligand complex, obtained through molecular docking, were chosen for the MD simulations to evaluate the system’s stability under solvent conditions. The final trajectory of equilibration was used as the initial structure for the data production simulation. We calculated the “root mean square deviation” (RMSD), and “root mean square fluctuations” (RMSF) of peptide-1 and peptide-2. The RMSD plot of the two peptides was shown in Fig 3. Starting from an initial value, the RMSD of peptide-1 showed a gradual increase during the course of the simulation. However, at 9.4 ns, the rate of increase accelerated significantly, ultimately reaching 5.18 Å by 14.7 ns. After that, the peptide-1 was stabilized in the active site of the visfatin during the simulation time of 50 ns (Fig 3A). The RMSD plot of peptide-2 showed the fluctuations up to 2ns after which the peptide gradually attained a stable position and large fluctuations were not observed during the simulation time (Fig 3B). 10.1371/journal.pone.0287577.g003 Fig 3 The RMSD plots of peptide-1 and -2 with respect to the initial complex during 50 ns of simulation time. (A) Peptide-1, (B) Peptide-2. The blue color trajectories represent the RMSD evolution of peptides. The RMSF plot was generated for two protein-peptides complexes to estimate the internal fluctuations in the protein and ligands. In the case of the peptide-1 complex, noticeable fluctuations were observed at residue positions 1 (0.5 nm) and 4 (0.42 nm) of the peptide, whereas, for peptide-2, no significant fluctuations were observed, with an RMSF value of less than 0.25 nm. According to the data presented in Fig 4A, a prominent fluctuation was observed in the visfatin chain at residue position 255, with a value of 0.55 nm. However, no significant fluctuations were observed for the remaining segments of the chain,, and the RMSF value remained below 0.25 nm. In the visfatin- peptide-2 complex (Fig 4B), no large fluctuations were observed and the RMSF value was less than 0.25 nm which meantthat both the visfatin and peptide were stable throughout the simulation time of 50 ns. 10.1371/journal.pone.0287577.g004 Fig 4 The RMSF plots of visfatin-peptide complexes. (A) Visfatin and peptide-1, (B) Visfatin and peptide-2. The black color trajectories represent the fluctuations in the visfatin and peptides. In silico toxicity, angiogenic, antihypertensive, and half-life predictions of peptides The in silico toxicity analysis showed that two peptides (peptide-1 and peptide-2) were non-toxic, with support vector machine (SVM) and quantitative matrix (QM) scores of lessthan zero (S1 and S2 Tables). The hemolytic prediction of these two peptides with an SVM score of zero represents non-hemolytic properties (S1 Table). The half-life of the designed peptides was less than 14 minutes, offering normal stability and negligible toxicity (S3 Table). These two peptides were non-anti-angiogenic and non-antihypertensive, with an SVM score of less than zero (S4 Table). Effect of the two peptides on cytotoxicity To investigate the cytotoxic effect of peptide-1 and -2, each peptide was measured in an MTT assay at concentrations of 0.1, 0.5, 1.0, and 2.0 μM. Treatment with the two peptides for 24 hours did not affect the viability of HUVECs (Fig 5). This indicated that peptide-1 and peptide-2 had no cytotoxic effect on HUVECs. 10.1371/journal.pone.0287577.g005 Fig 5 Cytotoxic effects of peptide-1 and -2 on HUVECs cells. Cell viability was estimated 24 hours after peptide-1 and -2 treatment using an MTT assay. Data are presented as mean±SD of three independent experiments. Vis: visfatin. Effects of the two peptides on angiogenesis The role of peptide-1 and -2 in promoting angiogenesis in vitro by invasion, migration, and tube formation in the HUVECs was examined at concentrations of 0.5 and 2.0 μM. As shown in Fig 4, cell invasion was significantly promoted more than two-fold by both peptides compared to the control group. Specifically, 0.5 μM peptide-1 and 2.0 μM peptide-2 increased cell invasion more than two-fold compared to the control and visfatin-treated groups. The increase in cell invasion by the peptides was higher than that by b-FGF in the positive control group (Fig 6). 10.1371/journal.pone.0287577.g006 Fig 6 Cytotoxic effects of peptide-1 and –2 on cell invasion. (A) Representative image and (B) graph of the invaded cells after 24 hours. HUVECs (2×104) were seeded in 100 μL of serum-free media with visfatin peptides added to the upper compartment of the transwell and the full medium was added to the lower compartment. After 24 hours, cell invasion was determined by counting the number of whole cell in a single filter using optical microscopy (40×). Data are presented as mean±SD of three independent experiments. ****P<0.0001 (vs control). Treatment with the two peptides resulted in a two-fold increase in cell migration at concentration of 0.5 μM for peptide-1 and 2.0 μM for peptide-2 compared to the control group (Fig 7). 10.1371/journal.pone.0287577.g007 Fig 7 Effects of peptide-1 and -2 on cell migration. (A) Representative image and (B) graph of the migrated cells at 18h. HUVECs (2×105/well) were seeded on 24-well plates and incubated overnight. Then, the cells were scratched using a P200 pipette tip and further incubated in media with or without peptide-1 and -2. Cells were allowed to migrate for 18h. Migration patterns were observed using a phase-contrast microscope (×40). Data are presented as mean±SD of three independent experiments. ****P<0.0001 (vs control). Angiogenic tube formation on Matrigel is an indication of the formation of blood vessels, such as capillaries, and is measured by two factors, mesh and master junction formation. The number of meshes was not significantly different when b-FGF and visfatin were used as positive controls compared to the control group, but was significantly increased by treatment with 0.5 μM concentration of peptide-1 and -2 (P<0.01, P<0.001, respectively). In contrast, the number of master junctions was significantly increased in both peptide-1 and -2 except at the 2.0 μM concentration of peptide-2. This increase was greater than that of b-FGF and visfatin (Fig 8). 10.1371/journal.pone.0287577.g008 Fig 8 Effect of peptide-1 and -2 on tube formation on Matrigel assays. (A) Representative tube formation images, (B) schematic image of angiogenesis, (C) number of branches, and (D) number of total branches. HUVECs (2×104/well) were seed on a layer of previously polymerized Matrigel and treated with or without peptide-1 and -2. The Matrigel culture was incubated at 37°C. After 4 hours, changes in cell morphology were captured using a phase-contrast microscope (×40). Each sample was assayed in duplicate, and independent experiments were repeated three times. *P<0.05, **P<0.01, and ***P<0.001 (vs control). Effects of the two peptides on the mRNA expression of VEGF and visfatin To confirm whether the two peptides stimulate the expression of visfatin and VEGF, the mRNA expressions of visfatin and VEGF were evaluated in the HUVECs treated with the two peptides. The expressions of both visfatin and VEGF-A were significantly increased at 0.5 μM of peptide-1 and at both 0.5 and 2.0 μM of peptide-2 compared to the control group (P<0.05). These expressions were comparable to those of visfatin and b-FGF treatment. Specifically, the VEGF-A expression was remarkably increased at 2.0 μM of peptide-2. On the other hand, 2.0 μM of peptide-1 did not increase the expression of visfatin and VEGF-A (Fig 9). 10.1371/journal.pone.0287577.g009 Fig 9 Effect of peptide-1 and -2 on expression of visfatin and VEGF-A. mRNA expressions of visfain and VEGF-A was examined in two peptides-treated cells by real-time quantitative PCR (RT-qPCR) analysis Each PCR was performed in triplicate for each sample. Relative gene expression levels were calculated versus GAPDH. Data are presented mean±SD. *P<0.05 and **P<0.01 (vs control). b-FGF: Basic fibroblast growth factor. Discussion This study showed that the two peptides derived from visfatin by protein-peptide docking simulations have more efficient angiogenic activity than visfatin itself. In addition, this study revealed that the two peptides stimulated the expression of VEGF-A and visfatin. This is the first report on the development of visfatin-derived peptides with better efficacy than the original full-length visfatin using computational techniques. It is well known that VEGF and periostin to stimulate angiogenesis. Therefore, some studies have reported the identification of VEGF- or periostin-derived angiogenic peptides containing about 10∼15 amino acids as agonists to overcome the disadvantages of using full-length proteins [48–50]. However, to our knowledge, no studies on visfatin-derived angiogenic peptides carried out to date. In addition, the amino acid sequences of peptide-1 and -2 identified in the present study have not been reported earlier and hence this study may be considered a novel study. The present study took into consideration two concepts to design visfatin agonist based on the active site. First, the designed peptide should act as a natural ligand of visfatin and produce similar biological effects when it binds to the receptor. Second, the designed peptide should be a visfatin mimetic. It was not possible to identify the protein-ligand binding structure using experimental methods because this would require considerable time and resource without the assistance of computer technology. However, as many computer-based docking simulation programs have been developed after the geometrical study of macromolecular-ligand interactions by Kuntz et al. [51], the study of protein-ligand interactions has now become very easy. Predicting the binding affinity between a ligand and a specific target protein is important in drug design. Molecular docking simulation is an excellent technique for identifying the optimal ligand-binding conformation for a specific target [52, 53]. For this purpose, two docking simulation programs, HADDOCK [37] and GalaxyPepDock [32] were used in this study. Through simulations, these two programs provide a score for the binding affinity between the ligand and the target protein or receptor. Among the 114 peptides obtained by the overlapping technique, peptides with an amino acid sequence suitable for the visfatin active site were evaluated using the HADDOCK docking simulation. The interaction between each peptide and visfatin was evaluated based on the similarity of the interaction between the ligand and the target protein stored in the GalaxyPepDock docking program. In this study, the overlapping technique was used prior to docking simulation to design the peptide. This strategy reduces the efforts required to synthesize a peptide and to analyze the interaction with the receptor. As a result, 114 peptides were generated from the truncated A-domain of viafatin and used in the docking simulation. The analysis of interactions between natural ligands and target proteins or receptors is very importantin peptide design [31, 54]. NMN is a natural ligand of visfatin that acts as an agonist [55]. This study simulated the docking of the natural NMN ligand (Fig 2) against visfatin to understand the catalytic site interactions. The results confirmed that the catalytic site interactions of nine peptides among those screened (Table 1) were similar to those of the natural NMN ligand. Since the biological unit of visfatin is dimer, it is also important to consider the dimer conformation for peptide design. Hence, the present study performed molecular docking simulation to design peptides by considering visfatin as a dimer in the GalaxyPepDock software and as a monomer in the HADDOCK software, and calculated their corresponding scores. As shown in the RMSD (Fig 3) and RMSF (Fig 4) plots, peptide-1 had more fluctuations than peptide 2. During the initial stages of the dynamics simulation, the N-terminus (residue position 1–4) of peptide 1 was weakly bound to visfatin, but the binding was disrupted at 9.4 ns. Subsequently, the structure of the N-terminus underwent changes and eventually reached a stable conformation after 14.7 ns. This resulted in fluctuations in the RMSF and RMSD plots. On the other hand, the C-terminus (residue position 6–10), which primarily interacts with the active site of visfatin, did not show significant structural changes during the simulation. Therefore, it appears that the structure of the visfatin-peptide1 complex is stable. In the case of peptide-2, the peptide chain was located inside the active site of visfatin, and fluctuated less during the simulation. Experimental determination of the toxicity of a large number of peptides is laborious, costly, and time-consuming. In silico toxicity analysis may provide an alternative to tedious experimental methods for predicting the toxicity of peptides. Based on our in silico toxicity predictions, out of the nine designed peptides, two peptides (peptide-1: LEYKLHDFGY and peptide-2; EYKLHDFGYRGV) were found to be non-toxic. In general, the half-life of an ideal peptide should be less than a few hours for it to stabilize and activate the immune system [56]. In this respect, these two peptides have normal stability and less toxicity with half-lives of less than 14 minutes. In addition, these two peptides were non-anti-angiogenic and non-antihypertensive. This study also evaluated the mRNA expression levels of visfatin to confirm the agonist activity of the designed peptides from in silico analysis. In addition, we evaluated the mRNA expression levels of VEGF and the angiogenesis efficacy of these peptides using in vitro analysis. In evaluating the angiogenesis efficacy of the two peptides (peptide-1 and -2), the determination of the treatment concentration also required careful consideration. Many previous studies had used concentrations in the range from 0.1 to 25 μM in the evaluation of angiogenesis activity of designed peptides [57, 58]. The present study carried out cytotoxicity assay in vitro at concentration range from 0.5 to 2.0 μM, and no cytotoxicity was observed at these concentrations. Based on these results, the lowest concentration of the peptides, at which no cytotoxicity was observed, was used to evaluate angiogenesis efficacy. One of the limitations of the present study is that it is uncertain whether the synthesized peptide had a configuration similar to visfatin as the same set of amino acids also had different configurations. Hence, we additionally evaluated the conformation and configuration of the synthetic peptides using circular dichroism (CD) spectroscopy. As shown in S1 Fig, peptide-1 was found to display two peaks (around 200 nm and 225 nm), and peptide-2 displayed a slight peak around 225 nm. These CD results did not show a typical spectrum of a-helix, b-sheet, and disordered form and therefore, we could not evaluate the configuration of the peptides. Nevertheless, the synthesized peptides may contain an optical isomer with the same configuration as that found in natural visfatin, and should have similar biological activities. In the future study, it would be important to consider the peptide configuration when designing peptides for therapeutic applications. Conclusions In conclusion, this study identified and developed two peptides as visfatin agonists, which stimulated the mRNA expression levels of visfatin and VEGF using in silico and in vitro analysis. Most importantly, these two peptides exhibited superior angiogenesis activity compared to visfatin itself. The peptides introduced in this study were developed for the first time and hold promise for facilitating the creation of therapeutic agents aimed at promoting skin regeneration and enhancing ovarian function. Supporting information S1 Table Toxicity and hemolytic activity predictions of designed peptides in different models using the SVM method. (DOCX) Click here for additional data file. S2 Table Toxicity predictions of designed peptides in different models using the QM method. (DOCX) Click here for additional data file. S3 Table Half-life of Peptides (HLP) in intestine-like environment and blood (Seconds). (DOCX) Click here for additional data file. S4 Table Angiogenic and hypertensive activities of designed peptides. (DOCX) Click here for additional data file. S1 Fig Circular dichroism (CD) spectroscopyof peptide-1 (A) and peptide-2 (B). (TIF) Click here for additional data file. 10.1371/journal.pone.0287577.r001 Decision Letter 0 Sever Belgin Academic Editor © 2023 Belgin Sever 2023 Belgin Sever https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version0 5 May 2023 PONE-D-23-06800Computer simulation approach to the identification of peptides with angiogenic activity based on the visfatin active sitePLOS ONE Dear Dr. Joo, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 19 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Belgin Sever, Ph.D. Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MSIT) (No. 2020R1A2C2014089)” We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “NO” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ********** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: After careful examination of this manuscript entitled " Computer simulation approach to the identification of peptides with angiogenic activity based on the visfatin active site" written by Choi et.al, I observed that this manuscript unsuitable to publish in this journal. Comments: 1. There was no novelty in this study. 2. MD simulation should be required for docking validation 3. Result and dicsussion section was not clear insight. 4. Results were not consistent with previous studies. 5. The biological unit of visfatin is dimer and why author did not consider dimer conformation for peptide design? Reviewer #2: In the manuscript (PONE-D-23-06800), authors researched the visfatin-derived angiogenic peptides by computer simulation approach. Overall, the topic is of scientific interest and would be of interest in the field of bioactive peptides. I think the manuscript can be accepted and published in PLOS ONE after a major revision. Suggestions are provided below: (1) Title: Should be “Computer simulation approach to the identification of visfatin-derived angiogenic peptides” rather “Computer simulation approach to the identification of peptides with angiogenic activity based on the visfatin active site”. (2) Keywords: Small pPeptide is not a standard term. I think that “oligopeptide” might be better than “small peptides”. (3) Section Abstract --Line 34: Should be “Firstly” rather “First”. --Line 35: Should be “was” rather “was first”. --Line 36: Should be “Secondly” rather “Second”. (4) Section Introduction: The introduction provides an adequate background on the topic. --Line 79-81: Authors are advised to add references. [1] Sridhar K, Inbaraj BS, Chen BH. Recent developments on production, purification and biological activity of marine peptides. Food Res Int. 2021, 147, 110468. [2] Suo SK, Zheng SL, Chi CF, Luo HY, Wang B. Novel angiotensin-converting enzyme inhibitory peptides from tuna byproducts-milts: Preparation, characterization, molecular docking study, and antioxidant function on H2O2-damaged human umbilical vein endothelial cells. Front. Nutr. 2022, 9, 957778. [3] Sheng Y, Wang WY, Wu MF, Wang YM, Zhu WY, Chi CF, Wang B. Eighteen Novel Bioactive Peptides from Monkfish (Lophius litulon) Swim Bladders: Production, Identification, Antioxidant Activity, and Stability. Mar Drugs. 2023, 21(3), 169. --Line 81-85: Authors are advised to add references. [1] Goginenia V, Hamannb MT. Marine natural product peptides with therapeutic potential: Chemistry, biosynthesis, and pharmacology. BBA - General Subjects 2018, 1862, 81-196. [2] Islam Md S, Wang H, Admassu H, Sulieman AA, Wei FA. Health benefits of bioactive peptides produced from muscle proteins: Antioxidant, anti-cancer, and anti-diabetic activities. Process Biochemistry 2022, 116, 116–125 [3] Kong J, Hu XM, Cai WW, Wang YM, Chi CF, Wang B. Bioactive peptides from Skipjack tuna cardiac arterial bulbs (II): Protective function on UVB-irradiated HaCaT cells through antioxidant and anti-apoptotic mechanisms. Marine Drugs, 2023, 21(2), 105. (5) Section Materials and Methods The materials and methods involved are generally detailed and the processes are clear. --However, in it is recommended that authors add references to experimental methods in this section. -- The authors seem to have left out the materials and reagents. It is recommended that the author fill in the reagent contents, which are very important for the reader to repeat the experiment. (6) Section Results I find the results very complete, clear, and concise. However, How can the authors determine whether the synthesized peptide is the same compound as the same amino acid sequence in visfatin? Because amino acids also have different configurations. (7) Section Discussion --Line 315-320: This sentence is repeated with the paragraph in the Introduction (Line 81-87). -- It is suggested that the results of this experiment should be compared with those of known peptides or other compounds with angiogenic activity. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ********** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0287577.r002 Author response to Decision Letter 0 Submission Version1 6 Jun 2023 Response to Editor-in-Chief Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: No Reviewer #2: Yes � We thank you for this observation. We asked a native speaker of English to review our manuscript for syntax and grammatical errors and we have corrected typographical errors. Response to reviewer #1 comments 1. There was no novelty in this study. � We thank you for this observation. Vascular endothelial growth factor (VEGF) and periostin have been well known to stimulate angiogenesis. However, some studies have demonstrated that the use of these full-length proteins has several inherent disadvantages, such as immunogenicity, lower stability, and loss of bioactivity (Lee et al., 2021). Therefore, some studies have reported the identification of VEGF- or periostin-derived angiogenic peptides containing about 10∼15 amino acids as an agonist to overcome the disadvantages of using full-length proteins (D’Andrea et al., 2005. Diana et al., 2008; Kim et al., 2017). However, to our knowledge, no studies on visfatin-derived angiogenic peptides have been carried out to date. In addition, the amino acid sequences of peptide-1 and -2 identified in the present study have not been reported in other studies. For this reason, we consider this to be a novel. Unfortunately, the reviewer felt that there was no novelty in this study, but if he can provide his logical reasoning for the same or previous studies, I will attempt to answer his concerns sincerely to his satisfaction. Lee K, Silva EA, Mooney DJ. Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface. 2011; 8(55): 153–170. https://doi:10.1098/rsif.2010.0223. D’Andrea LD, Iaccarino G, Fattorusso R, Sorriento D, Carannante C, Capasso D, et al. Targeting angiogenesis: Structural characterization and biological properties of a de novo engineered VEGF mimicking peptide. Proc Natl Acad Sci USA. 2005; 102 (40): 14215–14220. https:// doi: 10.1073/ pnas.0505047102. Diana D, Ziaco B, Colombo G, Scarabelli G, Romanelli A, Pedone C, Fattorusso R, et al. Structural determinants of the unusual helix stability of a de novo engineered vascular endothelial growth factor (VEGF) mimicking peptide. Chemistry 2008, 14(14): 4164–4166. https:// doi: 10.1002/chem.200800180. Kim BR, Kwon YW, Park GT, Choi EJ, Seo JK, Jang IH, et al. Identification of a novel angiogenic peptide from periostin. PLoS One. 2017 Nov 2;12(11):e0187464. https:// doi: 10.1371/journal.pone.0187464. 2. MD simulation should be required for docking validation � Thank you for pointing this out. We have considered this suggestion and performed the molecular dynamics simulations of visfatin-peptide complexes. We have included the required MD data in the materials and methods, results, and discussions sections of the revised manuscript. 3. Result and discussion section was not clear insight. � Thank you for pointing this out. We have revised the results and discussion section to clarify the insight from the study. 4. Results were not consistent with previous studies. � We are grateful for this comment. However, like our present study, few studies have developed short length amino acid sequences (around 10) with angiogenic activity derived from the active site of the original full-sized visfatin using computer simulation technique. The previous studies have all isolated full-sized natural proteins or peptides from natural source or using recombinant DNA technology etc. Furthermore, the amino acid sequences of peptide-1 and peptide-2 in this study are very unique, and they have been first developed by this study, to the best of our knowledge. So, we could not compare our results with the results of previous studies. The reviewer has stated that the results were not consistent with previous studies. I would like to assure the reviewer that I will attempt to sincerely address the concern if details of such earlier studies are provided. . 5. The biological unit of visfatin is dimer and why author did not consider dimer conformation for peptide design? � Thank you for pointing this out. We have performed molecular docking simulation to design peptides by considering the visfatin as a dimer in the GalaxyPepDock software and as a monomer in the HADDOCK software and calculated their corresponding scores. The highest-scoring two peptides obtained from the two methods above were considered for experimental analysis. Response to reviewer #2 comments: Reviewer #2: In the manuscript (PONE-D-23-06800), authors researched the visfatin-derived angiogenic peptides by computer simulation approach. Overall, the topic is of scientific interest and would be of interest in the field of bioactive peptides. I think the manuscript can be accepted and published in PLOS ONE after a major revision. Suggestions are provided below: (1) Title: Should be “Computer simulation approach to the identification of visfatin-derived angiogenic peptides” rather “Computer simulation approach to the identification of peptides with angiogenic activity based on the visfatin active site”. � Thank you for the suggestion. We have revised the title as suggested. (2) Keywords: Small peptide is not a standard term. I think that “oligopeptide” might be better than “small peptides”. � Thank you for pointing this out. We have made the change as suggested. (3) Section Abstract --Line 34: Should be “Firstly” rather “First”. --Line 35: Should be “was” rather “was first”. --Line 36: Should be “Secondly” rather “Second”. � We are grateful for this comment. We have made the change to the above. (4) Section Introduction: The introduction provides an adequate background on the topic. --Line 79-81: Authors are advised to add references. --Line 81-85: Authors are advised to add references. � We thank you for this comment. We have changed this part to provide references as recommended as follows: However, high molecular weight of visfatin is a major limitation to its development as a therapeutic drug. In contrast, therapeutic peptides consisting of short-length amino acid sequence (3 to 20) have exhibited beneficial effects for the treatment of several health conditions (Lau et al., 2018; Wang et al., 2022). The peptides offer specific advantages such as good efficacy, good safety, low immunogenicity, high membrane permeability, and low cost compared to therapeutic proteins and antibodies (Fosgerau & Hoffmann, 2015; Muttenthaler 2021; Wang et al., 2022). For this reason, interest in the field of therapeutic peptides, including marine peptides, has increased in recent years. To date, more than 170 peptides are in active clinical development with many more in the preclinical stages (Research, 2016; Sridhar et al., 2021; Wang et al., 2022; Sheng et al., 2023). In particular, recent improvements in peptide screening and computational biology have increased the demand for peptide drug discovery (Wang et al., 2022). Lau JL, Dunn M. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorg Med Chem. 2018; 26(10): 2700-2707. https://doi: 10.1016/j.bmc.2017.06.052. Wang L, Wang N, Zhang W, Cheng X, Yan Z, Shao G, et al. Therapeutic peptides: current applications and future directions. Signal Transduct Target Ther. 2022; 7(1): 48. https://doi: 10.1038/s41392-022-00904-4. Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discov Today. 2015; 20(1): 122-128. https://doi: 10.1016/j.drudis.2014.10.003. Muttenthaler M, King GF, Adams DJ, Alewood PF. Trends in peptide drug discovery. Nat Rev Drug Disco. 2021; 20(4): 309–325. https://doi: 10.1038/s41573-020-00135-8. Research TM. Global industry analysis, size, share, growth, trends and forecast. Pept. Mark. 2016–2024, 2016. Sridhar K, Inbaraj BS, Chen BH. Recent developments on production, purification and biological activity of marine peptides. Food Res Int. 2021; 147: 110468. https://doi: 10.1016/j.foodres.2021.110468. Sheng Y, Wang WY, Wu MF, Wang YM, Zhu WY, Chi CF, Wang B. Eighteen novel bioactive peptides from Monkfish (Lophius litulon) Swim Bladders: production, identification, antioxidant activity, and stability. Mar Drugs. 2023; 21(3): 169. https:// doi: 10.3390/md21030169. (5) Section Materials and Methods The materials and methods involved are generally detailed and the processes are clear. -- The authors seem to have left out the materials and reagents. It is recommended that the author fill in the reagent contents, which are very important for the reader to repeat the experiment. � We thank you for observation. We have changed several parts in the Materials and Method section to comply with your recommendation. (6) Section Results I find the results very complete, clear, and concise. However, How can the authors determine whether the synthesized peptide is the same compound as the same amino acid sequence in visfatin? Because amino acids also have different configurations. � One of the limitations of the present study is that it is uncertain whether the synthesized peptide had a configuration similar to visfatin as the same set of amino acids also had different configurations. Hence, we additionally evaluated the conformation and configuration of the synthetic peptides using circular dichroism (CD) spectroscopy. As shown in Fig. S1, peptide-1 was found to display two peaks (around 200 nm and 225 nm), and peptide-2 displayed a slight peak around 225 nm. These CD results did not show a typical spectrum of a-helix, b-sheet, and disordered form and therefore, we could not evaluate the configuration of the peptides. Nevertheless, the synthesized peptides may contain an optical isomer with the same configuration as that found in natural visfatin, and should have similar biological activities. In the future study, it would be important to consider the peptide configuration when designing peptides for therapeutic applications. (7) Section Discussion --Line 315-320: This sentence is repeated with the paragraph in the Introduction (Line 81-87). � We thank you for this observation. We have deleted the concerned sentence -- It is suggested that the results of this experiment should be compared with those of known peptides or other compounds with angiogenic activity. � We are grateful for this comment. We have introduced the following section in the discussion. It is well known that VEGF and periostin to stimulate angiogenesis. Therefore, some studies have reported the identification of VEGF- or periostin-derived angiogenic peptides containing about 10∼15 amino acids as agonists to overcome the disadvantages of using full-length proteins (D’Andrea et al., 2005. Diana et al., 2008; Kim et al., 2017). However, to our knowledge, no studies on visfatin-derived angiogenic peptides carried out to date. In addition, the amino acid sequences of peptide-1 and -2 identified in the present study have not been reported earlier and hence this study may be considered a novel study. D’Andrea LD, Iaccarino G, Fattorusso R, Sorriento D, Carannante C, Capasso D, et al. Targeting angiogenesis: Structural characterization and biological properties of a de novo engineered VEGF mimicking peptide. Proc Natl Acad Sci USA. 2005; 102 (40): 14215–14220. https:// doi: 10.1073/ pnas.0505047102. Diana D, Ziaco B, Colombo G, Scarabelli G, Romanelli A, Pedone C, Fattorusso R, et al. Structural determinants of the unusual helix stability of a de novo engineered vascular endothelial growth factor (VEGF) mimicking peptide. Chemistry 2008, 14(14): 4164–4166. https:// doi: 10.1002/chem.200800180. Kim BR, Kwon YW, Park GT, Choi EJ, Seo JK, Jang IH, et al. Identification of a novel angiogenic peptide from periostin. PLoS One. 2017 Nov 2;12(11):e0187464. https:// doi: 10.1371/journal.pone.0187464. Attachment Submitted filename: Response to Reviewers.docx Click here for additional data file. 10.1371/journal.pone.0287577.r003 Decision Letter 1 Sever Belgin Academic Editor © 2023 Belgin Sever 2023 Belgin Sever https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version1 9 Jun 2023 Computer simulation approach to the identification of visfatin-derived angiogenic peptides PONE-D-23-06800R1 Dear Dr. Joo, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Belgin Sever, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: 10.1371/journal.pone.0287577.r004 Acceptance letter Sever Belgin Academic Editor © 2023 Belgin Sever 2023 Belgin Sever https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 14 Jun 2023 PONE-D-23-06800R1 Computer simulation approach to the identification of visfatin-derived angiogenic peptides Dear Dr. Joo: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Assoc. Prof. Dr. Belgin Sever Academic Editor PLOS ONE ==== Refs References 1 Hoeben A , Landuyt B , Highley MS , Wildiers H , Van Oosterom AT , De Bruijn EA . Vascular endothelial growth factor and angiogenesis. Pharmacol Rev. 2004; 56 (4 ): 549–580. doi: 10.1124/pr.56.4.3 15602010 2 Tonnesen MG , Feng X , Clark RA . Angiogenesis in wound healing. J Invest Dermatol Symp Proc. 2000; 5 (1 ): 40–46. doi: 10.1046/j.1087-0024.2000.00014.x 11147674 3 Li J , Zhang YP , Kirsner RS . Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix. Microsc Res Tech 2003; 60 (1 ): 107–114. doi: 10.1002/jemt.10249 12500267 4 Geva E , Jaffe RB . Role of vascular endothelial growth factor in ovarian physiology and pathology. Fertil Steril. 2000; 74 (3 ): 429–438. doi: 10.1016/s0015-0282(00)00670-1 10973633 5 Devesa J , Caicedo D . The Role of growth hormone on ovarian functioning and ovarian angiogenesis. Front Endocrinol. (Lausanne). 2019, 10 : 450. doi: 10.3389/fendo.2019.00450 31379735 6 Billhaq DH , Lee SH , Lee S . The potential function of endometrial-secreted factors for endometrium remodeling during the estrous cycle. Anim Sci J. 2020; 91 (1 ): e13333. doi: 10.1111/asj.13333 31909524 7 Ruth KS , Day FR , Hussain J , Martínez-Marchal A , Aiken CE , Azad A , et al . Genetic insights into biological mechanisms governing human ovarian ageing. Nature. 2021; 596 (7872 ): 393–397. doi: 10.1038/s41586-021-03779-7 34349265 8 Kriebs A. Genetics of female reproductive longevity. Nat Rev Endocrinol. 2021; 17 (10 ): 575. doi: 10.1038/s41574-021-00558-2 34408296 9 Shimazu T , Jiang JY , Liijima K , Miyabayashi K , Oogawa Y , Sasada H . Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature glits. Biol Reprod. 2003; 69 (4 ): 388–393. doi: 10.1095/biolreprod.103.016311 12826586 10 Danforth DR , Arbogast LK , Ghosh S , Dickerman A , Rofagha R , Friedman CI . Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary. Biol Reprod. 2003; 68 (5 ): 1736–1741. doi: 10.1095/biolreprod.101.000679 12606430 11 Sills ES , Wood SH . Autologous activated platelet-rich plasma injection into adult human ovary tissue: molecular mechanism, 162 analysis, and discussion of reproductive response. Biosci Rep. 2019; 39 (6 ): BSR20190805. doi: 10.1042/BSR20190805 31092698 12 Hsu CC , Hsu L , Hsu I , Chiu YJ , Dorjee S . Live birth in woman with premature ovarian insufficiency receiving ovarian 231 administration of platelet-rich plasma (PRP) in combination with gonadotropin: A case report. Front Endocrinol (Lausanne). 2020; 11 : 50. doi: 10.3389/fendo.2020.00050 32140135 13 Choi KH , Joo BS , Sun SD , Park MJ , Son JB , Joo JK , et al . Administration of visfatin during superovulation improves developmental competency of oocytes and fertility potential in aged female mice. Fertil Steril. 2012; 97 (5 ): 1234–1241.e3. doi: 10.1016/j.fertnstert.2012.02.032 22425197 14 Park BK , Park MJ , Kim HG , Han SE , Kim CW , Joo BS , et al . Role of visfatin in restoration of ovarian aging and fertility in the mouse aged 18 months. Reprod Sci. 2020; 27 (2 ): 681–689. doi: 10.1007/s43032-019-00074-9 31942708 15 Samal B , Sun Y , Stearns G , Xie C , Suggs S , McNiece I . Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol. 1994; 14 (2 ): 1431–1437. doi: 10.1128/mcb.14.2.1431-1437.1994 8289818 16 Ognjanovic S , Bao S , Yamamoto SY , Garibay-Tupas J , Samal B , Bryant-Greenwood GD . Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes. J Mol Endocrinol. 2001; 26 (2 ): 107–117. doi: 10.1677/jme.0.0260107 11241162 17 Hug C , Lodish HF . Medicine. Visfatin: a new adipokine. Science. 2005; 307 (5708 ): 366–367. doi: 10.1126/science.1106933 15604359 18 Curat CA , Wegner V , Sengenes C , Miranville A , Tonus C , Busse R , et al . Macrophages in human visceral adipose tissue: increased accumulation in obesity and a source of resistin and visfatin. Diabetologia. 2006; 49 (4 ): 744–747. doi: 10.1007/s00125-006-0173-z 16496121 19 Kim SR , Bae SK , Choi KS , Park SY , Jun HO , Lee JY , et al . Visfatin promotes angiogenesis by activation of extracellular signal-regulated kinase 1/2. Biochem Biophys Res Commun. 2007; 357 (1 ): 150–156. doi: 10.1016/j.bbrc.2007.03.105 17408594 20 Adya R , Tan BK , Punn A , Chen J , Randeva HS . Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc Res. 2008; 78 (2 ): 356–365. doi: 10.1093/cvr/cvm111 18093986 21 Xiao J , Xiao ZJ , Liu ZG , Gong HY , Yuan Q , Wang S , et al . Involvement of dimethylarginine dimethylaminohydrolase-2 in visfatin-enhanced angiogenic function of endothelial cells. Diabetes Metab Res. 2009; 25 (3 ): 242–249. doi: 10.1002/dmrr.939 19229883 22 Bae YH , Bae MK , Kim SR , Lee JH , Wee HJ , Bae SK . Up-regulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis. Biochem Biophys Res Commun. 2009; 379 (2 ): 206–211. doi: 10.1016/j.bbrc.2008.12.042 19100714 23 Lee K , Silva EA , Mooney DJ . Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface. 2011; 8 (55 ): 153–170. doi: 10.1098/rsif.2010.0223 20719768 24 Lau JL , Dunn M . Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorg Med Chem. 2018; 26 (10 ): 2700–2707. doi: 10.1016/j.bmc.2017.06.052 28720325 25 Wang L , Wang N , Zhang W , Cheng X , Yan Z , Shao G , et al . Therapeutic peptides: current applications and future directions. Signal Transduct Target Ther. 2022; 7 (1 ): 48. doi: 10.1038/s41392-022-00904-4 35165272 26 Fosgerau K , Hoffmann T . Peptide therapeutics: current status and future directions. Drug Discov Today. 2015; 20 (1 ): 122–128. doi: 10.1016/j.drudis.2014.10.003 25450771 27 Muttenthaler M , King GF , Adams DJ , Alewood PF . Trends in peptide drug discovery. Nat Rev Drug Disco. 2021; 20 (4 ): 309–325. doi: 10.1038/s41573-020-00135-8 33536635 28 Research TM . Global industry analysis, size, share, growth, trends and forecast. Pept. Mark. 2016–2024 , 2016. 29 Sridhar K , Inbaraj BS , Chen BH . Recent developments on production, purification and biological activity of marine peptides. Food Res Int. 2021; 147 : 110468. doi: 10.1016/j.foodres.2021.110468 34399466 30 Sheng Y , Wang WY , Wu MF , Wang YM , Zhu WY , Chi CF , et al . Eighteen novel bioactive peptides from Monkfish (Lophius litulon) Swim Bladders: production, identification, antioxidant activity, and stability. Mar Drugs. 2023; 21 (3 ): 169. doi: 10.3390/md21030169 36976218 31 Hruby V. Designing peptide receptor agonists and antagonists. Nat Rev Drug Discov. 2002; 1 (11 ): 847–858. doi: 10.1038/nrd939 12415245 32 Lee H , Heo L , Lee MS , Seok C . Galaxy PepDock: a protein-peptide docking tool based on interaction similarity and energy optimization. Nucleic Acids Res. 2015; 43 (W1 ): W431–435. doi: 10.1093/nar/gkv495 25969449 33 Kim MK , Lee JH , Kim H , Park SJ , Kim SH , Kang GB , et al . Crystal structure of visfatin/pre-B cell colony-enhancing factor-1/nicotinamide phosphoribosyl transferase, free and in complex with the anti-cancer agent FK-866. J Mol Biol. 2006; 362 (1 ): 66–77. doi: 10.1016/j.jmb.2006.06.082 16901503 34 Zhang LQ , Heruth DP , Ye SQ . Nicotinamide phosphoribosyltransferase in human diseases. J Bioanal Biomed. 2011; 3 : 13–25. doi: 10.4172/1948-593X.1000038 22140607 35 Kaur H , Garg A , Raghava GPS . PEPstr: A de novo method for tertiary structure prediction of small bioactive peptides. Protein Pept Lett. 2007; 14 (7 ): 626–630. doi: 10.2174/092986607781483859 17897087 36 Singh S , Singh H , Tuknait A , Chaudhary K , Singh B , Kumaran S . PEPstrMOD: structure prediction of peptides containing natural, non-natural and modified residues. Biol Direct. 2015; 10 : 73. doi: 10.1186/s13062-015-0103-4 26690490 37 Dominguez C , Boelens R , Bonvin AMJ . HADDOCK: a protein−protein docking approach based on biochemical or biophysical information. J Am Chem Soc. 2003; 125 (7 ): 1731–1737. doi: 10.1021/ja026939x 12580598 38 Tian C , Kasavajhala K , Belfon KA , Raguette L , Huang H , Migues AN , et al . f19SB: Amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. J Chem Theory Comput. 2019; 16 (1 ): 528–552. doi: 10.1021/acs.jctc.9b00591 31714766 39 Izadi S , Anandakrishnan R , Onufriev AV . Building water models: a different approach. J Phys Chem Lett. 2014; 5 (21 ): 3863–3871. doi: 10.1021/jz501780a 25400877 40 Jo S , Kim T , lyer VG , Im W . CHARMM‐GUI: a web‐based graphical user interface for CHARMM. J Comput Chem. 2008; 29 (11 ): 1859–1865. doi: 10.1002/jcc.20945 18351591 41 Abraham MJ , Murtola T , Schulz R , Páll S , Smith JC , Hess B , et al . GROMACS: High-performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015; 1–2 : 19–25. doi: 10.1016/j.softx.2015.06.001 42 Gupta S , Kapoor P , Chaudhary K , Gautam A , Kumar R . In silico approach for predicting toxicity of peptides and proteins. PLoS ONE 2013; 8 (9 ): e73957. doi: 10.1371/journal.pone.0073957 24058508 43 Chaudhary K , Kumar R , Singh S , Tuknait A , Gautam A , Mathur D , et al . A web server and mobile app for computing hemolytic potency of peptides. Sci Rep. 2016; 6 : 22843. doi: 10.1038/srep22843 26953092 44 Mathur D , Singh S , Mehta A , Agrawal P , Raghava G . In silico approaches for predicting the half-life of natural and modified peptides in blood. PLoS One. 2018;13 (6 ): e0196829. doi: 10.1371/journal.pone.0196829 29856745 45 Sharma A , Singla D , Rashid M , Gajendra PSR . Designing of peptides with desired half-life in intestine-like environment. BMC Bioinformatics. 2014; 15 (1 ): 282. doi: 10.1186/1471-2105-15-282 25141912 46 Kumar R , Chaudhary K , Chauhan JS , Nagpal G , Kumar R , Sharma M , et al . An in silico platform for predicting, screening and designing of antihypertensive peptides. Sci Rep. 2015; 5 : 12512. doi: 10.1038/srep12512 26213115 47 Ettayapuram Ramaprasad AS , Singh S , Gajendra PSR , Venkatesan S . AntiAngioPred: A server for prediction of anti-angiogenic peptides. PLos One. 2015; 10 (9 ): e0136990. doi: 10.1371/journal.pone.0136990 26335203 48 D’Andrea LD , Iaccarino G , Fattorusso R , Sorriento D , Carannante C , Capasso D , et al . Targeting angiogenesis: Structural characterization and biological properties of a de novo engineered VEGF mimicking peptide. Proc Natl Acad Sci USA. 2005; 102 (40 ): 14215–14220. doi: 10.1073/pnas.0505047102 16186493 49 Diana D , Ziaco B , Colombo G , Scarabelli G , Romanelli A , Pedone C , et al . Structural determinants of the unusual helix stability of a de novo engineered vascular endothelial growth factor (VEGF) mimicking peptide. Chemistry 2008, 14 (14 ): 4164–4166. doi: 10.1002/chem.200800180 18384035 50 Kim BR , Kwon YW , Park GT , Choi EJ , Seo JK , Jang IH , et al . Identification of a novel angiogenic peptide from periostin. PLoS One. 2017 Nov 2;12 (11 ):e0187464. doi: 10.1371/journal.pone.0187464 29095886 51 Kuntz ID , Blaney JM , Oatley SJ , Langridge R , Ferrin TE . A geometric approach to macromolecule-ligand interactions. J Mol Biol. 1982; 161 (2 ): 269–88. doi: 10.1016/0022-2836(82)90153-x 7154081 52 Ferreira LG , Dos Santos RN , Oliva G , Andricopulo AD . Molecular docking and structure-based drug design strategies. Molecules. 2015; 20 (7 ): 13384–13421. doi: 10.3390/molecules200713384 26205061 53 Dos Santos RN , Ferreira LG , Andricopulo AD . Practices in molecular docking and structure-based virtual screening. Methods Mol Biol. 2018; 1762 : 31–50. doi: 10.1007/978-1-4939-7756-7_3 29594766 54 Jakubowski H . Agonist and Antagonist of Ligand Binding to Receptors. 2021 Aug 17. In LibreTexts Biology. Available from: https://bio.libretexts.org. 55 Sommer G , Garten A , Petzold S , Beck-Sickinger AG , Blüher M , Stumvoll M , et al . Visfatin/PBEF/Nampt: structure, regulation and potential function of a novel adipokine. Clin Sci (Lond). 2008; 115 (1 ): 13–23. doi: 10.1042/CS20070226 19016657 56 Zafar S , Bai B , Guo J , Muhammad SA , Naqvi STQ , Shabbir MN , et al . Experimental study of potential CD8+ trivalent synthetic peptides for liver cancer vaccine development using sprague dawley rat models. Biomed Res Inter. 2022; 2002 : 4792374. doi: 10.1155/2022/4792374 35686237 57 Griffioen AW , van der Schaft DW , Barendsz-Janson AF , Cox A , Struijker Boudier HA , Hillen HF , et al . Anginex, a designed peptide that inhibits angiogenesis. Biochem J. 2001; 354 (Pt 2 ): 233–242. doi: 10.1042/0264-6021:3540233 11171099 58 Li J , Post M , Volk R , Gao Y , Li M , Metais C , et al . PR39, a peptide regulator of angiogenesis. Nat Med 2000; 6 (1 ): 49–55. doi: 10.1038/71527 10613823