Peptides with biological properties, that is, bioactive peptides, are a class of biomolecules whose health-promoting properties are increasingly being exploited in food and health products. However, research on targeted techniques for the detection and quantification of these peptides is still in its infancy. Such information is needed in order to enhance the biological and chemometric characterization of peptides and their subsequent application in the functional food and pharmaceutical industries. In this review, the role of classic techniques such as electrophoretic, chromatographic, and peptide mass spectrometry in the structure-informed detection and quantitation of bioactive peptides are discussed. Prospects for the use of aptamers in the characterization of bioactive peptides are also discussed. PRACTICAL APPLICATIONS: Although bioactive peptides have huge potential applications in the functional foods and health area, there are limited techniques in enhancing throughput detection, quantification, and characterization of these peptides. This review discusses state-of-the-art techniques relevant in complementing bioactive detection and profiling irrespective of the small number of amino acid units. Insights into challenges, possible remedies and prevailing areas requiring thorough research in the extant literature for food chemists and biotechnologists are also presented.
This study explores the inhibitory properties of camel whey protein hydrolysates (CWPH) toward α-amylase (AAM) and α-glucosidase (AG). A general full factorial design (3 × 3) was applied to study the effect of temperature (30, 37, and 45°C), time (120, 240, and 360 min), and enzyme (pepsin) concentration (E%; 0.5, 1, and 2%). The results showed that maximum degree of hydrolysis was obtained when hydrolysis was carried out at higher temperature (45°C; P < 0.05), compared with lower temperatures of 30 and 37°C. Electrophoretic pattern displays degradation of all protein bands upon hydrolysis by pepsin at various hydrolysis conditions applied. All the 27 CWPH generated showed significant AAM and AG inhibitory potential as indicated by their lower IC50 values (mg/mL) compared with intact whey proteins. In total 196 peptides were identified from selected hydrolysates and 15 potential peptides (PepSite score > 0.8; http://pepsite2.russelllab.org/) were explored via in silico approach. Novel peptides PAGNFLMNGLMHR, PAVACCLPPLPCHM, MLPLMLPFTMGY, and PAGNFLPPVAAAPVM were identified as potential inhibitors for both AAM and AG due to their high number of binding sites and highest binding probability toward the target enzymes. CCGM and MFE, as well as FCCLGPVPP were identified as AG and AAM inhibitory peptides, respectively. This is the first study that reports novel AG and AAM inhibitory peptides from camel whey proteins. The future direction for this research involves synthesis of these potential AG and AAM inhibitory peptides in a pure form and investigate their antidiabetic properties in the in vitro, as well as in vivo models. Thus, CWPH can be considered for potential applications in glycaemic regulation.
In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
Recent evidence suggests that the formation of soluble amyloid β (Aβ) aggregates with high toxicity, such as oligomers and protofibrils, is a key event that causes Alzheimer's disease (AD). However, understanding the pathophysiological role of such soluble Aβ aggregates in the brain in vivo could be difficult due to the lack of a clinically available method to detect, visualize, and quantify soluble Aβ aggregates in the brain. We had synthesized a novel fluorinated curcumin derivative with a fixed keto form, named as Shiga-Y51, which exhibited high selectivity to Aβ oligomers in vitro. In this study, we investigated the in vivo detection of Aβ oligomers by fluorine-19 (19F) magnetic resonance imaging (MRI) using Shiga-Y51 in an APP/PS1 double transgenic mouse model of AD. Significantly high levels of 19F signals were detected in the upper forebrain region of APP/PS1 mice compared with wild-type mice. Moreover, the highest levels of Aβ oligomers were detected in the upper forebrain region of APP/PS1 mice in enzyme-linked immunosorbent assay. These findings suggested that 19F-MRI using Shiga-Y51 detected Aβ oligomers in the in vivo brain. Therefore, 19F-MRI using Shiga-Y51 with a 7 T MR scanner could be a powerful tool for imaging Aβ oligomers in the brain.
Camel milk proteins are an important substrate for bioactive peptides generation. This study investigates in-vitro antidiabetic effect (via inhibition of α-amylase (AA), α-glucosidase (AG) and dipeptidyl peptidase IV (DPP-IV)) of bovine (BC) and camel casein (CC) hydrolysates. Further, effect of simulated gastrointestinal digestion (SGID) on inhibitory potential of generated hydrolysates was also explored. Both BC and CC hydrolysates displayed potent inhibitory properties against AA (IC50 value- 0.58 & 0.59 mg/mL), AG (IC50 value- 1.04 & 0.59 mg/mL) and DPP-IV (IC50 value- 0.62 & 0.66 mg/mL), respectively. Among different peptides identified in BC and CC hydrolysates, it was observed that FLWPEYGAL was predicted to be most potent inhibitory peptide against AA. While LPTGWLM, MFE and GPAHCLL as most active inhibitor of AG and HLPGRG, QNVLPLH and PLMLP were predicted to be active against DPP-IV. Overall, BC and CC hydrolysates can be proposed to be used in different food formulations as functional antidiabetic agents.
Keratins are a group of important proteins in skin and hair and as biomaterials they can provide desirable properties such as strength, biocompatibility, and moisture regaining and retaining. The aim of this work is to develop water-soluble keratin polypeptides from sheep wool and then explore how their surface adsorption behaves with and without surfactants. Successful preparation of keratin samples was demonstrated by identification of the key components from gel electrophoresis and the reproducible production of gram scale samples with and without SDS (sodium dodecylsulphate) during wool fibre dissolution. SDS micelles could reduce the formation of disulphide bonds between keratins during extraction, reducing inter-molecular crosslinking and improving keratin polypeptide solubility. However, Zeta potential measurements of the two polypeptide batches demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing complete removal of SDS during purification by dialysis. In spite of different solubility from the two batches of keratin samples prepared, very similar adsorption and aggregation behavior was revealed from surface tension measurements and dynamic light scattering. Mixing of keratin polypeptides with SDS and C12TAB (dodecyltrimethylammonium bromide) led to the formation of keratin-surfactant complexes that were substantially more effective at reducing surface tension than the polypeptides alone, showing great promise in the delivery of keratin polypeptides via the surface active complexes. Neutron reflection measurements revealed the coexistence of surfactant and keratin polypeptides at the interface, thus providing the structural support to the observed surface tension changes associated with the formation of the surface active complexes.
PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed.
METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry.
RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05).
CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Protein arginine deiminase type IV (PAD4) is responsible for the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullinated protein is the autoantigen in rheumatoid arthritis, and therefore, PAD4 is currently a promising therapeutic target for the disease. Recently, we reported the importance of the furan ring in the structure of PAD4 inhibitors. In this study, the furan ring was incorporated into peptides to act as the "warhead" of the inhibitors for PAD4. IC50 studies showed that the furan-containing peptide-based inhibitors were able to inhibit PAD4 to a better extent than the furan-containing small molecules that were previously reported. The best peptide-based inhibitor inhibited PAD4 reversibly and competitively with an IC50 value of 243.2 ± 2.4 μm. NMR spectroscopy and NMR-restrained molecular dynamic simulations revealed that the peptide-based inhibitor had a random structure. Molecular docking studies showed that the peptide-based inhibitor entered the binding site and interacted with the essential amino acids involved in the catalytic activity. The peptide-based inhibitor could be further developed into a therapeutic drug for rheumatoid arthritis.
We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg)12. While it is possible that the palmitoyl-cyclic-(D-Arg)12/HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg)12/HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg)12/HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg)12/HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg)12/HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg)12/HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis.
Several bioactive molecules isolated from the saliva of blood-sucking arthropods, such as mosquitoes, have been shown to exhibit potential anticoagulant function. We have previously identified a 30kDa allergen named Aegyptin-like protein (alALP), which is highly homologous to Aegyptin, from the salivary glands of female Aedes albopictus (Asian tiger mosquito). In this study, we identified the conserved functional domain of alALP by using bioinformatic tools, and expressed the His-tagged alALP recombinant protein in sf9 insect cells by generation and transfection of a baculoviral expression plasmid carrying the fulllength cDNA of alALP. We purified this recombinant protein and examined its function on the inhibition of blood coagulation. The results showed that the purified His-alALP prolonged the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) in vitro as well as the Bleeding Time (BT) in vivo, which suggest that alALP could be a novel anticoagulant.
Matched MeSH terms: Salivary Proteins and Peptides/chemistry*
Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.
Tuberculosis (TB) presently accounts for high global mortality and morbidity rates, despite the introduction four decades ago of the affordable and efficient four-drugs (isoniazid, rifampicin, pyrazinamide and ethambutol). Thus, a strong need exists for new drugs with special structures and uncommon modes of action to effectively overcome M. tuberculosis. Within this scope, antimicrobial peptides (AMPs), which are small, cationic and amphipathic peptides that comprise a section of the innate immune system, are currently the leading potential agents for the treatment of TB. Many studies have recently illustrated the capability of anti-mycobacterial peptides to disrupt the normal mycobacterial cell wall function through various modes, thereby interacting with the intracellular targets, as well as encompassing nucleic acids, enzymes and organelles. This review presents a wide array of antimicrobial activities, alongside the associated properties of the AMPs that could be utilized as potential agents in therapeutic tactics for TB treatment.
The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity.
Stone fish is an under-utilized sea cucumber with many health benefits. Hydrolysates with strong ACE-inhibitory effects were generated from stone fish protein under the optimum conditions of hydrolysis using bromelain and fractionated based on hydrophobicity and isoelectric properties of the constituent peptides. Five novel peptide sequences with molecular weight (mw) < 1000 daltons (Da) were identified using LC-MS/MS. The peptides including Ala-Leu-Gly-Pro-Gln-Phe-Tyr (794.44 Da), Lys-Val-Pro-Pro-Lys-Ala (638.88 Da), Leu-Ala-Pro-Pro-Thr-Met (628.85 Da), Glu-Val-Leu-Ile-Gln (600.77 Da) and Glu-His-Pro-Val-Leu (593.74 Da) were evaluated for ACE-inhibitory activity and showed IC50 values of 0.012 mM, 0.980 mM, 1.310 mM, 1.440 mM and 1.680 mM, respectively. The ACE-inhibitory effects of the peptides were further verified using molecular docking study. The docking results demonstrated that the peptides exhibit their effect mainly via hydrogen and electrostatic bond interactions with ACE. These findings provide evidence about stone fish as a valuable source of raw materials for the manufacture of antihypertensive peptides that can be incorporated to enhance therapeutic relevance and commercial significance of formulated functional foods.
Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142-156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41-55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.
This study investigates the complete molecular characterization including bioinformatics characterization, gene expression, synthesis of N and C terminal peptides and their antimicrobial activity of the core histone 4 (H4) from freshwater giant prawn Macrobrachium rosenbergii (Mr). A cDNA encoding MrH4 was identified from the constructed cDNA library of M. rosenbergii during screening and the sequence was obtained using internal sequencing primers. The MrH4 coding region possesses a polypeptide of 103 amino acids with a calculated molecular weight of 11kDa and an isoelectric point of 11.5. The bioinformatics analysis showed that the MrH4 polypeptide contains a H4 signature at (15)GAKRH(19). Multiple sequence alignment of MrH4 showed that the N-terminal (21-42) and C-terminal (87-101) antimicrobial peptide regions and the pentapeptide or H4 signature (15-19) are highly conserved including in humans. The phylogenetic tree formed two separate clades of vertebrate and invertebrate H4, wherein MrH4 was located within the arthropod monophyletic clade of invertebrate H4 groups. Three-dimensional model of MrH4 was established using I-TASSER program and the model was validated using Ramachandran plot analysis. Schiffer-Edmundson helical wheel modeling was used to predict the helix propensity of N (21-42) and C (87-101) terminal derived Mr peptides. The highest gene expression was observed in gills and is induced by viral [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacterial (Aeromonas hydrophila and Vibrio harveyi) infections. The N and C terminal peptides were synthesized and their antimicrobial and hemolytic properties were examined. Both peptides showed activity against the tested Gram negative and Gram positive bacteria; however, the highest activity was noticed against Gram negative bacteria. Among the two peptides used in this study, C-terminal peptide yielded better results than the N-terminal peptide. Therefore, C terminal peptide can be recommended for the development of an antimicrobial agent.
Prolonged chewing of betel quid is known to cause oral diseases, including cancer. The present study was performed to screen for aberrant proteins in the saliva of habitual betel quid chewers compared to nonchewers. Saliva of female subjects (n = 10) who had been chewing betel quid for more than 20 years and nonbetel quid chewers (n = 10) of the same gender and range of age was analyzed by gel-based proteomics. Increased structural microheterogeneity of saliva haptoglobin beta chains indicated by shifts of focused spots similar to that earlier reported in patients with oral squamous cell carcinoma, and their relatively higher abundance compared to nonbetel quid chewers, were detected in saliva protein profiles of all chewers. In addition, the majority of the betel quid chewers also showed significant higher abundance of hemopexin, alpha-1B glycoprotein, alpha1-antitrypsin, complement C3, and transthyretin. These proteins had previously been associated with several different cancers. Our data demonstrated different forms of protein aberration in the saliva of betel quid chewers, which may be indicative of early oral precancerous conditions.
Matched MeSH terms: Salivary Proteins and Peptides/chemistry
Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients' 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients' saliva α1-antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α-1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.
Matched MeSH terms: Salivary Proteins and Peptides/chemistry
Photodynamic therapy (PDT) is a cancer treatment modality that requires three components, namely light, dioxygen and a photosensitizing agent. After light excitation, the photosensitizer (PS) in its excited state transfers its energy to oxygen, which leads to photooxidation reactions. In order to improve the selectivity of the treatment, research has focused on the design of PS covalently attached to a tumor-targeting moiety. In this paper, we describe the synthesis and the physico-chemical and photophysical properties of six new peptide-conjugated photosensitizers designed for targeting the neuropilin-1 (NRP-1) receptor. We chose a TPC (5-(4-carboxyphenyl)-10,15, 20-triphenyl chlorine as photosensitizer, coupled via three different spacers (aminohexanoic acid, 1-amino-3,6-dioxaoctanoic acid, and 1-amino-9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid) to two different peptides (DKPPR and TKPRR). The affinity towards the NRP-1 receptor of the conjugated chlorins was evaluated along with in vitro and in vivo stability levels. The tissue concentration of the TPC-conjugates in animal model shows good distribution, especially for the DKPPR conjugates. The novel peptide-PS conjugates proposed in this study were proven to have potential to be further developed as future NRP-1 targeting photodynamic therapy agent.
Vascular endothelial growth factor (VEGF) and its co-receptor neuropilin-1 (NRP-1) are important targets of many pro-angiogenic factors. In this study, nine peptides were synthesized and evaluated for their molecular interaction with NRP-1 and compared to our previous peptide ATWLPPR. Docking study showed that the investigated peptides shared the same binding region as shown by tuftsin known to bind selectively to NRP-1. Four pentapeptides (DKPPR, DKPRR, TKPPR and TKPRR) and a hexapeptide CDKPRR demonstrated good inhibitory activity against NRP-1. In contrast, peptides having arginine residue at sites other than the C-terminus exhibited low activity towards NRP-1 and this is confirmed by their inability to displace the VEGF165 binding to NRP-1. Docking study also revealed that replacement of carboxyl to amide group at the C-terminal arginine of the peptide did not affect significantly the binding interaction to NRP-1. However, the molecular affinity study showed that these peptides have marked reduction in the activity against NRP-1. Pentapeptides having C-terminal arginine showed strong interaction and good inhibitory activity with NRP thus may be a good template for anti-angiogenic targeting agent.