International Journal of Peptide Research and Therapeutics

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Online ISSN: 1573-3904
Print ISSN: 1573-3149
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Importance of naturally occurring antimicrobial compounds from fish mucus
  • Ahmed Hussain
    Ahmed Hussain
  • Shashwati Ghosh Sachan
    Shashwati Ghosh Sachan
Microbes are helpful and destructive to human health and other living organisms. Microbes can be eliminated by using antibiotics against them, but their capability to resist regularly encountering antibiotics makes them more injurious. Microbes can adjust and adapt according to the chemicals used against them and become antibiotic resistant. Thus, the requirement for novel antimicrobial compounds increases with time to treat antibiotic-resistant microbes. Fish epidermal mucus encounters various pathogens present in their surrounding environment. It has become a rich source of novel antimicrobial compounds mainly antimicrobial peptides that can be used against various antibiotic-resistant pathogenic microbes. Compounds extracted from epidermal mucus can be used synergistically with other antibiotics or resistance modifying agents to inhibit the growth of resistant microbes. Fishes are consumed as a protein-rich food source worldwide and contribute to the world economy. Diseases in fish cause significant losses in the economic benefits exploited by fishermen and industries based on fisheries products. This paper will review compounds from fish epidermal mucus and their use to control the growth of antibiotic-resistant or non-resistant pathogenic microbes of humans and fishes. So, to increase fisheries’ economic benefits and decrease infections involving resistant microbes.
This study aimed to investigate the effect of two synthetic peptides, HL-7 and HL-10, extracted from the venom of H. lepturus, on insulin secretion and glucose absorption. The INS-1 cell line was used to measure the amount of insulin secretion in the culture medium, while the HSkMC cell line was applied to analyze the amount of glucose uptake and GLUT4, AMP and Akt gene expression. Also, streptozotocin-induced diabetic rats were used to assess plasma glucose, insulin concentrations, and GLUT4 gene expression levels in soleus muscle isolated from rats. Finally, the in-silico analysis evaluated the interaction of the two peptides with the transcription factor NF-κB. The results showed that the amount of insulin released and glucose uptake by INS-1 and HSkMC cells treated with HL-7 and HL-10 peptides significantly increased (p < 0.0001). Accordingly, both peptides decreased plasma glucose and elevated insulin levels in diabetic rats compared to untreated diabetic rats (p < 0.0001). Also, an increase was found in GLUT4 gene expression at both mRNA and protein levels in peptide-treated cells compared to untreated cells. Also, an increase in GLUT4, AMPK and Akt gene expression was observed at both mRNA and protein levels in peptide-treated cells compared to untreated cells. The in-silico results demonstrated strong hydrogen bonds between the two peptides with the NF-κB protein, indicating the possible inhibitory role of these peptides against NF-κB. The results of the present study showed that the two peptides can control diabetes and glucose homeostasis in a glucose-dependent and -independent manner. Also, one of the reasons for increased glucose uptake can be due to the regulation of key upstream signaling kinases such as AMPK and Akt, which ultimately leads to the regulation of GLUT4 expression and facilitating glucose uptake by peptide-treated HSkMC cells. Since the analyzed synthetic peptides had no significant toxicity against INS-1 and HSkMC cells, they could be suitable candidates for humans with diabetes in clinical trials.
In the current study, we evaluated the effect of leucine-enkephalin (L-ENK) on lipid deposition and GSK-3β/mTOR signaling in zebrafish. Zebrafish were exposed with 2.5, 12.5, and 25 µg/g body weight (bw) L-ENK for four weeks. Our results demonstrated that 2.5, 12.5, and 25 µg/g L-ENK enhanced the hepatic gene expression and activities of fatty acid synthesis enzymes. The level of triglyceride (TG) and the number of lipid droplet was increased by 2.5, 12.5, and 25 µg/g L-ENK treatments. Moreover, 2.5, 12.5, and 25 µg/g L-ENK elevated mTOR and PPARγ gene expression, but inhibited GSK-3β and TSC2 expression in the zebrafish liver. Our results showed that L-ENK induces fatty acid synthesis and lipid deposition, and the signaling GSK-3β/mTOR may be involved in this regulatory process.
A Insulin receptor extracellular ectodomain (in ribbon representation with one IR protomer in magenta and the second in coral) with two insulin molecules (in surface representation) bound in Sites 1 and 1’, and two insulins bound in Sites 2 and 2’. Drawn based on the 6SOF.pdb cryo-EM structure (Gutmann et al. 2020). B Extracellular domain of IGF-1 receptor with one IGF-1 molecule bound to Site 1. Potential alternative Site 1’ is also labeled. Drawn based on the 6PYH.pdb cryo-EM structure (Li et al. 2019). The color scheme is the same as in A
Schematic representation of IGF-1 Dimers 1–3 synthesized and tested in this study. IGF-1 monomers are interconnected between C-terminal Ala⁷⁰ of the N-terminal monomer and N-terminal Gly¹ of the C-terminal monomer with Ser-Gly chains of different length. All three constructs were prepared with an additional Gly⁻¹ residue (at the position − 1) on the N-terminus. IGF-1 peptide chains are shown as blue lines, with disulfide bridges in yellow and terminal residues in black. Amino acids of the connecting linkers are shown in red
A RP-HPLC purification of Dimer 1 products. B RP-HPLC purification of Dimer 2 products. C RP-HPLC purification of Dimer 3 products. The m/z signals of compounds (labeled as e.g., Dimer 1a obtained from peak a in A etc.) are shown in Table 1
Comparison of the CD spectra of Dimers 2c and 2d with the native IGF-1 (A) and the corresponding secondary structure content for native IGF-1 (B), Dimer 2c (C), and Dimer 2d (D)
Insulin-like growth factor 1 (IGF-1) and its IGF-1 receptor (IGF-1R) belong to an important biological system that is involved in the regulation of normal growth, but that has also been recognized as playing a role in cancer. IGF-1R antagonists could be interesting for the testing of their potential antiproliferative properties as an alternative to IGF-1R tyrosine-kinase inhibitors or anti-IGF-1R monoclonal antibodies. In this study, we were inspired by the successful development of insulin dimers capable of antagonizing insulin effects on the insulin receptor (IR) by simultaneous binding to two separated binding sites and by blocking structural rearrangement of the IR. We designed and produced in Escherichia coli three different IGF-1 dimers in which IGF-1 monomers are interlinked through their N- and C-termini, with linkers having 8, 15 or 25 amino acids. We found that the recombinant products were susceptible to the formation of misfolded or reduced variants, but that some of them were able to bind IGF-1R in low nanomolar affinities and all of them activate IGF-1R proportionally to their binding affinities. Overall, our work can be considered as a pilot study that, although it did not lead to the discovery of new IGF-1R antagonists, explored the possibility of recombinant production of IGF-1 dimers and led to the preparation of active compounds. This work could inspire further studies dealing, for example, with the preparation of IGF-1 conjugates with specific proteins for the study of the hormone and its receptor or for therapeutic applications. Graphical Abstract
Figure depicting the various risk factors associated with the pathogenesis of breast cancer
Figure depicting few of the chemical structures of therapeutic peptides
Pictorial representation of various anticancer peptides and their role to be used as targeted therapy in breast cancer
Picture representing the stages of breast cancer with the possible role of therapeutic peptides in suppressing the progression of breast cancer
Breast cancer is among the most frequently occurring cancer for women globally. Conventional therapies for breast cancer are found to be expensive and cause severe aftereffects; further increasing drug resistance is also attracting the interest of researchers to explore novel therapeutic strategies to treat breast cancer. Among the cutting-edge approaches, several therapeutic peptides that target and destroy tumour cells have attracted much concern. Short chains of amino acids known as therapeutic peptides have a high affinity for binding and regulating a specific protein interaction. A reliable way to get around the issues with other therapies is through targeted therapy. Additionally, it enables medications to selectively localize at predetermined locations. Therapeutic peptides are an innovative and promising method for treating solid tumours, notably breast cancer. The aim of this review article is to give an extensive and informative overview of therapeutic peptides and how they are used to treat breast cancer. Research into new, efficient therapeutic approaches for breast cancer treatment can be supported by the development of beneficial therapeutic technologies, like therapeutic peptides and their different anti-cancerous potential against tumor cells. Graphical Abstract
Melittin, the most potent pharmacological ingredient of honey bee venom, induces haemolysis, lymphocyte lysis, long-term pain, localised inflammation, and hyperalgesia. In this study, efforts were made to subdue the melittin’s ill effects using a chaperone peptide called ‘mini-αA-crystallin’ (MAC) derived from eye lens αA-crystallin. Haemolytic test on human red blood cells, percentage viability, and DNA diffusion assay on Human peripheral blood lymphocytes (HPBLs) were performed with melittin in the presence or absence of MAC. Propidium iodide and Annexin V-FITC dual staining were performed to analyse quantitative levels of necrotic and apoptotic induction by melittin in the presence or absence of MAC on HPBLs using a flow cytometer. A computational study to find out the interactions between MAC and melittin was undertaken by modelling the structure of MAC using a PEP-FOLD server. The result showed that MAC inhibited melittin-induced lysis in nucleated (lymphocytes) and enucleated (RBC) cells. Flow cytometric analysis revealed a substantial increase in the necrotic and late apoptotic cells after treating HPBLs with melittin (4 µg/ml) for 24 h. Treatment with MAC at a 2:1 molar ratio prevented HPBLs from developing melittin-induced necrosis and late apoptosis. In the docking study, hydrogen, van der Waals, π-π stacking, and salt bridges were observed between the MAC and melittin complex, confirming a strong interaction between them. The MAC-melittin complex was stable during molecular dynamics simulation. These findings may be beneficial in developing a medication for treating severe cases of honeybee stings.
The synthesized cyclic homodimer and grafted versions of LyP-1 peptide. The cyclic backbones are shown with thick lines, whereas disulfide bonds are shown with thin lines. SFTI-1 was not synthesized
Serum stability of LyP-1 peptides. Cyclic homodimer LyP-1 peptide has the highest serum stability among the designed peptides in %100 serum. LyP-1-loop 1-SFTI-1 peptide was completely degraded in serum in four hours, whereas other grafted peptides had better stability than LyP-1-loop 1-SFTI-1
Anticancer peptide therapeutics are promising candidates for cancer treatment as they are highly specific to their targets and can have tumor-homing abilities. Several studies have been shown that a nine-residue tumor-homing LyP-1 peptide can be used in anticancer agent development and in tumor imaging studies. However, because it is a small linear peptide, it is very unstable in serum as most of the peptide therapeutics face before they enter to clinical studies. The main aim of this study is to enhance the serum stability of LyP-1 using cyclization and grafting (and/or retro grafting) strategies. Four grafted versions onto sunflower trypsin inhibitor peptide SFTI-1 and a cyclic homodimerized version of LyP-1 were synthesized and tested on MDA-MB-435S cancer (melanoma) cells and HFF-1 normal fibroblast cells in comparison with cisplatin chemotherapeutics. The cyclic homodimer LyP-1 peptide exhibited extraordinary stability in serum. Additionally, the retro grafted peptides have also improved stability that indicates the retro sequence concept provides peptides with higher stability.
Scorpion venom contains various peptides that could be utilized to treat various diseases, including cancer. This study aimed to evaluate the anti-cancer activity of scorpion venom peptide (Smp24) using a solid Ehrlich Carcinoma (SEC) mice model. SEC model was established by subcutaneous transplantation of SEC cells into Swiss albino female mice afterward subcutaneous injection of the Smp24 peptide compared to 5-Fluorouracil (5-FU) as a standard drug. Various biochemical, hematological, histopathological, immunohistochemical, and molecular (western blotting and RT-PCR) assays were performed to evaluate the antitumor activity of Smp24. Results revealed that Smp24 peptide significantly reduced tumor volume. Interestingly, Smp24 peptide significantly restored normal body functions in cancer-treated groups by maintaining HB, RBC’s, and WBC’s levels, reducing the elevated serum ALT and AST, and increasing total protein and albumin as well as enhancing antioxidant status through reducing the level of MDA and NO and elevating GSH, SOD, and CAT levels. Moreover, it restored the normal morphology of the liver and kidney tissues and improved hematological parameters in cancer-treated animals. Smp24 induced apoptosis in SEC cells, through upregulation of caspase-3 and BAX and the downregulation of VEGF, Bcl-2, p53, PCNA, and Ki67. Moreover, results exhibited the apoptotic and antiangiogenic effects of Smp24 against SEC cancer cells. These findings supported our previous results about the anti-cancer efficacy of Smp24 and made it a good candidate for developing effective and safe anti-cancer agents.
AMPs have various mechanisms of actions. A AMPs can form pores in the cell membrane, leading to disruption of ionic gradients and cell death. Pore-formation models are carpet model, barrel stave model, toroidal model, and detergent model (Wimley 2010) B AMPs can bind to different intracellular targets (DNA, RNA, and proteins) and damage essential cellular mechanisms by inhibition of DNA or RNA synthesis and/or enzymatic activities (Brogden 2005). C AMPs can trigger pro-inflammatory effects through the chemotaxis of immune cells, including neutrophils, monocytes, CD4⁺ T-cells, CD8⁺ T-cells, and dendritic cells. Moreover, they can exert anti-inflammatory effects via inhibition of cellular responses to IFN-γ, which suppresses the secretion of proinflammatory cytokines (Raheem and Straus 2019)
Four main modes of action by which AMPs exert their anti-cancer properties
Four subclasses of AMPs based on secondary structures. A magainin-2 (α-helical AMP, PDB ID: 2MAG), B defensin-5 (β-sheet AMP, PDB ID: 2LXZ), C indolicidin (extended AMP, PDB ID: 1QXQ), and D microcin-J25 (loop AMP, PDB ID: 1S7P). Hydrophobic regions have been depicted on the surfaces of peptides. The surface potential was depicted on a scale from − 3 (blue, low hydrophobic regions) to + 3 (red, high hydrophobic regions). Figures were generated using Discovery Studio v 3.5
Antimicrobial peptides (AMPs) are part of the innate immune system of various species. In addition to extensive studies on their probable application as antibacterial and antifungal antibiotics, AMPs are also under investigation for their wound healing, anti-inflammatory, and immunomodulatory effects. Furthermore, the functionality and advantages of AMPs in the design and production of anticancer agents have been assessed. In this regard naturally occurring AMPs or their synthetic derivatives, have been verified for their cytotoxicity on normal and cancer cells. Moreover, some AMPs (or their derivatives) are shown to act as cancer-specific cell penetrating peptides or homing peptides, which makes them suitable for targeting a cytotoxic moiety or a drug carrier toward cancer cells. Therefore, considering the growing number of reports on the advantageous properties of AMPs in the field of anticancer agents, in this study we will review and summarize the corresponding in vitro and in vivo results of these peptides with dual antimicrobial and anticancer properties. To build up a more comprehensive manuscript, we will also discuss the molecular changes made to the natural structure of AMPs, making them suitable for anticancer applications.
Schematic representation of P. nigriventer δ-ctenitoxin-Pn2a containing five disulfide bridges, three of them forming the Cys knot motif (CI–CIV, CII–CV, CIII–CVI). The selected sequence (34-48) of the epitope is indicated in black
ESI mass spectra. a Ac-GYFWIAWYKLAN-Abu-KKG-NH2: [M + 3H]³⁺ m/z = 658.0, [M + 3H + 44]³⁺ m/z = 672.7, [M + 2H]²⁺ m/z = 986.5 and [M + 2H + 44]²⁺ m/z = 1008.5. b Palm-GYFWIAWYKLAN-Abu-KKG-NH2: [M + 3H]³⁺ m/z = 723.4, [M + 3H + 44]³⁺ m/z = 738.1 and [M + 2H]² m/z = 1084,6. c (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2: [M + 6H]⁶⁺ m/z = 685.9, [M + 6H + 44]⁶⁺ m/z = 693.2, [M + 5H]⁵⁺ m/z = 822.9, [M + 5H + 44]⁵⁺ m/z = 831.7, [M + 5H + 88]⁵⁺ m/z = 840.5, [M + 4H]⁴⁺ m/z = 1028.3, [M + 4H + 44]⁴⁺ m/z = 1039.3, [M + 4H + 88]⁴⁺ m/z = 1050.3, [M + 3H]³⁺ m/z = 1370.8, [M + 3H + 44]³⁺ m/z = 1385.4). M + 44 and M + 88 corresponded to the incomplete decarboxylation intermediate Trp(CO2H) during Trp(Boc) final cleavage
RP-HPLC analysis of the synthesized peptides in a XSelect Peptide CSH reverse-phase C18 column (130 Å × 3.5 μm × 5 μm) at 50 °C, at a flow rate of 1.6 mL/min and execute in a Shimadzu LC-20AT instrument, Photodiode Array UV–Vis Detector (SPD-M20A). Solvent A: 0.045% TFA in H2O. Solvent B: 0.036% TFA in acetonitrile. The right axes indicate % of solution B in solution A (%B) in the gradient program. a Ac-GYFWIAWYKLAN-Abu-KKG-NH2, b Palm-GYFWIAWYKLAN-Abu-KKG-NH2, c (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2
Cell viability MTT assay. Histograms represent cell viability expressed as percentage of the control after exposure to 0.00, 0.25, 0.50, 1.00, 2.50, 5.00, and 10.00 μM of peptide Ac-GYFWIAWYKLAN-Abu-KKG-NH2 (a), Palm-GYFWIAWYKLAN-Abu-KKG-NH2 (b) and (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (c). Data show the mean ± SD (n = 3). Statistically significant difference vs. control according to One-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 compared with the control group (0.00 μM)
NF-κB cellular distribution detected by immunofluorescence (green) in the RAW 264.7 macrophage cell line. Nuclei were stained with Hoechst (blue). Control cells (a–ai) and cells treated for 4 h with 0.5 µM of: lipopolysaccharide (b–bi), Ac-GYFWIAWYKLAN-Abu-KKG-NH2 (c–ci), Palm-GYFWIAWYKLAN-Abu-KKG-NH2 (d–di) and (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (e–ei). Arrows indicate INF-κB immunolabeling in macrophage nuclei. Bar scale 10 µm
The venom toxin δ-ctenitoxin-Pn2a of the spider Phoneutria nigriventer can cause severe envenomation in humans. Furthermore, the cystine-knot motif of δ-ctenitoxin-Pn2a provides exceptional stability, thereby hampering immune response activation. Here we identified epitope G³⁴YFWIAWYKLANCKK⁴⁸ from δ-ctenitoxin-Pn2a through the Immune Epitope Database Analysis Resource and used it to design antigenic peptides. The Cys residue was replaced by α-aminobutyric acid (Abu) to prevent disulfide bond formation. To increase the immunogenicity of these molecules, branched and N-palmitoylated versions were synthesized. Ac-GYFWIAWYKLAN-Abu-KKG-NH2 (A), Palm-GYFWIAWYKLAN-Abu-KKG-NH2 (B) and (Ac-GYFWIAWYKLAN-Abu-KK)2-KG-NH2 (C) were prepared by solid-phase synthesis and their identity was confirmed by ESI–MS. They were then studied by RP-HPLC and all the chromatograms obtained showed only one main peak. Cytotoxicity was evaluated on the murine macrophage cell line RAW 264.7 using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay in the presence of increasing doses of each peptide (0.25–10.0 µM). Peptide A did not show cytotoxicity between 0.25 and 10.0 µM, while B and C did at concentrations equal or over 0.5 and 10.0 respectively. The cellular distribution of NF-κB was examined by immunofluorescence after exposing macrophages to 0.5 µM of each peptide. Early activation was observed for all three peptides, thereby indicating that they are promising immunogens for antivenom production. Nevertheless, in vivo tests are still required to assess their immunogenic capacity and whether the antibodies generated can confer protection against the venom.
Representative schematic of the immobilization of peptides on metallic surfaces by silanization without crosslinking molecules and plasma polymerization
Representative scheme of PEG electrodeposition for immobilization of bioactive peptides. In an electrochemical cell, the PEG solution containing the functional groups is used as the electrolyte. In the electrodeposition process, the metallic material is coated with PEG and subsequently immersed in a peptide solution. Based on Park et al. (2011)
Details of the plasma polymerization method on the metallic surface
The literature is rich in research that seeks strategies capable of improving the antimicrobial activity, antibiofilm and tissue interaction of metallic materials for biomedical purposes. Bioactive peptides appear as good candidates in the development of new biomaterials, since they are short molecules that have a wide range of biological activities. However, their use can be difficult given the susceptibility to degradation associated with the environment in which they interact. As a consequence, choosing the appropriate immobilization methodology is crucial to ensure the effectiveness of a biomaterial. Here we consider works published in indexed journals from the 1990s to the present to present a mini methodological review on the strategies to carry out modifications of metallic surfaces with bioactive peptides, their advantages and disadvantages. It was possible to verify the recurrent use of metallic materials such as titanium, cobalt, chromium and stainless steels for a multitude of applications in the biomedical area. With regard to bioactive peptides, the interest in those capable of increasing osseointegration and in those that can confer antimicrobial action and/or anti-biofilm formation is notable. Despite this, advances regarding the mechanism of action of immobilized peptides and their impact on the corrosion properties of materials are still needed.
Effects of 5-min reperfusion with analogues A1 and A3 on functional and metabolic parameters of isolated working rat heart at the end of reperfusion (from Pisarenko et al. 2015a). (A) LVDP x HR product; (B) cardiac output; (C) myocardial phosphocreatine; (D) myocardial lactate; (E) LV diastolic pressure at the end of reperfusion; (F) LDH release for 5-min reperfusion after global ischaemia. C- control, 5-min Langendorff perfusion with STH2 at 25 °C before 40 min normothermic global ischaemia followed by 5-min Langendorff reperfusion with Krebs–Henseleit buffer (KHB) at 37 °C before 25-min reperfusion with KHB at 37 °C according to Neely. A, A1, A3—the same protocol with 5-min Langendorff reperfusion with KHB containing 140 μM apelin-12 (A), analogue 1 (A1) or analogue 3 (A3) at 37 °C after global ischaemia. Values are the means ± standard error of the mean of 10–12 experiments. *p < 0.05 versus control
A Signaling mechanisms involved in cardioprotection afforded by postconditioning with analogue 1. B, C Inhibitors of the RISK pathway reduce beneficial effects of analogue 1 on myocardial infarct size (IS/AAR,%,) and plasma activity of CK-MB in rats in vivo at the end of reperfusion (from Pisarenko et al. 2015c). steady state, C control; A1 analogue1, UO UO126, an inhibitor of MEK1/2; LY, LY294002, a PI3K inhibitor; N, L-NAME, a NOS inhibitor. Values are the means ± standard error of the mean of 8 experiments.*p < 0.05 vs. control; #p < 0.05 vs. A1
The apelin/APJ system is involved in many physiological functions and pathophysiological effects in cardiovascular diseases, making it a promising drug target. This narrative review briefly summarizes data on experimental conditioning of the heart with modified structural analogues of apelin-12. Apelin-12 analogues resistant to proteolytic cleavage in human plasma were synthetized by targeted substitution of amino acid residues in the structure of natural apelin-12 by an automated solid-phase method using Fmoc technology. These peptides are able to mimic the protective effect of apelin-12 in both ex vivo and in vivo models of ischaemia/reperfusion (I/R) myocardial injury. Intravenous administration of apelin-12 analogues at the onset of reperfusion reduces the size of acute myocardial infarction in rats, preserves the metabolic and antioxidant state of the area at risk during reperfusion, and improves cardiomyocyte membrane integrity. Postconditioning effects are mediated by signaling via PLC and survival kinases, PI3K and MEK1/2, with further activation of downstream targets, NO synthase and mitochondrial KATP channels, and sarcolemmal Na⁺/H⁺ and Na⁺/Ca²⁺ exchangers. Given the role of apelin/APJ in cardiovascular diseases, future perspectives of development of pharmacological postconditioning with apelin-12 analogues are discussed. This strategy may provide important therapeutic benefits in the treatment of cardiovascular disease.
Peptides are small polymers composed of 40 or fewer amino acids and are an increasingly important class of drugs. Therapeutic peptides are less immunogenic and more economical than biologics while offering greater safety, selectivity, efficacy, and specificity than small molecule drugs. Despite this, they are challenging to mold in a stable formulation due to their susceptibility to degradation. By understanding the degradation behavior of such peptide drugs, researchers and pharmaceutical manufacturers can design safe, effective, and stable peptide formulations. From a scientific standpoint, forced degradation studies are an indispensable tool to forecast the stability of any molecule during its development phase. Being structurally diverse, the degradation of peptide drugs is different from the small molecules. This review provides a practical summary of strategies adopted to perform the stress stability testing for different peptide therapeutics including a selection of stress conditions, degradation products formed, and an analytical methodology used for the characterization of degradation products. Hence, it will help in developing a protocol for performing forced degradation studies on peptide therapeutics of interest. In-depth discussions of the different peptide degradation mechanisms are also included, along with preventive measures. The information presented here in the form of case studies on the degradation profile of existing peptide drugs will help in designing more resistant peptide drugs by chemical modifications, as well as aid in the advancement of generic peptide drug product development. In brief, this review presents the way of controlling peptide degradation from synthesis to formulation development based on their constituted amino acids.
Heat absorption curves. Solid line—clinically healthy 22–45 year old individuals; gashed line—DBC patients
Metaphases with different number of associations: a—with two “closed” associations- 1(13:13; 13:14: 14:13) and 2(14:15;14:15); b—with four “open” associations- 3(13:15); 4(13:21);c -with one “closed” association 5(14:21;14:22;13:21;13:22) and two “open” associations 6(15:21); 7(13:14). Associations are indicated by arrows
The intensity of participation in association chromatids of chromosome—13, 14, 15, 21 and 22 in the individuals control group (22–45 yr.) and from patients with DBC and patients treated with LCo
The types of associations of homologous chromosome chromatids (13:13; 14:14; 15:15; 21:21 and 22:22) in cultures of patients with DBC, DBC cultures treated with LCo, and the control group (22-45 yr). *p < 0.05; **p < 0.001
After the completion of the Human Genome Project, the strategic direction of modern genetics shifted towards functional genomics, which also includes non-coding regions of DNA. Total non-coding DNA and satellite stalk with ribosomal cystrons, comprising repetitive sequences of nucleotides localized in heterochromatin. The functions of heterochromatin remain unclear. The present study aims to investigate of total heterochromatin and NOR heterochromatin in patients with DBC and with the bioregulator and a heavy metal (CoCl2) (LCo). The level of total heterochromatin by differential scanning microcalorimetry (DSM) was investigated. Intensity of ribosomal cystrons of chromatid satellite stalks (NOR heterochromatin) in 32 lymphocyte cultures from healthy individuals and the 18 from patients with DBC, and DBC with the bioregulator and a heavy metal also have been study. DSM showed the melting point of heterochromatin indicating slight chromatin heterochromatinization. The activity of ribosomal cystrons of chromatid on the 15 chromosomes included in associations in the control group was reduced compared to the activity of other chromatids (14 = 21 = 22 > 13 > 15). But, the chromatids 15 chromosome patients with DBC, compared to the control group had high activity inclusion in associations (p < 0.001). The activity ribosomal cystrons the type of association on chromosomes 15:15 in patients with DBC also was significantly increased (p < 0.001). However in patients with DBC + LCo, the activity ribosomal cystrons of chromatids on chromosome 15 and the frequency of association 15:15 was reduced (p < 0.001), which was mainly in line with the data of middle age. Our results indicated the differential activity of ribosomal cystrons of chromatids of chromosome 15 in patients with DBC (significantly increased) and DBC with the LCo (significantly reduced) in contrast to the control group. The study of acrocentric chromatids with epigenetic variability in the activity of ribosomal genes in pathology is a new direction in medicine, indicating the diagnosis of diseases and defining a new treatment strategy in the future.
Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.
Culture of ACL fibroblasts and determination of intervention dose. (A) Primary ACL fibroblasts. (B) Third-generation ACL fibroblasts. (C) Results of cck8 treated with different concentrations (0,5,10,20,40,60 ng/ml) of Ghrelin for different times (12,24,48,72 h). (D) Results of cck8 treated with different concentrations of TNF-α (0,5,10,20,40 ng/ml) for different times (24,48,72,120 h).Scale bars: 200 μm
Ghrelin promoted the migration of ACL fibroblasts. (A) Cell migration results in the control group. (B) Cell migration results in the TNF-α group. (C) Cell migration results in the TNF-α + Ghrelin group. (D) Comparison of cell migration results between groups. Scale bars: 200 μm. *, p < 0.05; **, p < 0.01;***, p < 0.001 compared to the control group
Ghrelin regulated the expression of pyroptosis-related molecules. (A) Representative western blot images of changes in the expression level of GSDMD, caspase-1, IL-1β, and IL-18. (B-E) protein expression of IL-1β, IL-18, caspase-1 and GSDMD. (F-I) mRNA expression of IL-1β, IL-18, caspase-1 and GSDMD in ACL fibroblasts. GAPDH was used as an internal control to calculate relative expression. *, p < 0.05; **, p < 0.01;***, p < 0.001 compared to the control group
Ghrelin regulated the expression of NF-κB p65/NLRP3 protein. (A) Representative western blot images of changes in the expression level of P-P65, NLRP3.(B) protein expression of P-P65. (C) protein expression of NLRP3. GAPDH was used as an internal control to calculate NLRP3 relative expression. P65 was used as an internal control to calculate P-P65 relative expression.*, p < 0.05; **, p < 0.01;***, p < 0.001 compared to the control group
Ghrelin promoted the repair of the injured ACL in vivo. (A) HE staining of ACL tissue. (B) Masson staining of ACL tissue. (C) Ghrelin improves pyroptosis in ACLI via the NF-κB p65/NLRP3 pathway. Scale bars: 200 μm
To explore whether Ghrelin could inhibit anterior cruciate ligament (ACL) derived fibroblasts pyroptosis and promote migration through regulating NF-κB p65/NLRP3 signaling. In this study, fibroblasts extracted from the ACL of clinical patients were collected for subsequent cellular experiments and to determine the model of TNF-α cell inflammation and the optimal concentration of Ghrelin intervention. An ACL partial injury model was established in Sprague Dawley rats given Ghrelin or Saline intervention. The repair of the ACL injury was determined by detecting the expression of pyroptosis molecules in ACL fibroblasts, changes in migration capacity, and histological staining. Cell migration was significantly inhibited in the TNF-α inflammation model group compared to the control group, and the expression of cellular pyroptosis proteins was significantly increased. Ghrelin intervention improved cell migration and inhibited the expression of pyroptosis proteins. Further study revealed that the expression of NF-κB p65/NLRP3 was also significantly reduced, suggesting that Ghrelin may inhibit the pyroptosis of fibroblasts by suppressing NF-κB p65/NLRP3. In Sprague Dawley rats with partial ACL injury, Ghrelin treatment for 4 or 8 weeks promoted ACL repair by increasing the number of fibroblasts and optimizing the distribution of collagen fibers compared to the injury group. Our study illustrates the ability of Ghrelin to inhibit pyroptosis and improve cell migration ability, thereby promoting repair of injured ACLs, which will provide a new strategy for the treatment of ACL injuries.
Angiotensin-I Converting Enzyme (ACE) plays a pivotal role in regulating hypertension. Inhibition of ACE by potent inhibitors is an excellent approach to treating high blood pressure and related cardiac ailments. Dietary proteins are a good source of bioactive peptides with a broad spectrum of pharmacological activities. An attempt has been made in this investigation to study the structure–activity relationship of a novel octapeptide ACE inhibitor having the amino acid sequence: isoleucine-glycine-proline-glycine-proline-phenylalanine-serine-arginine (IGPGPFSR; molecular weight 830.953 Dalton), isolated and identified from Lamellidens marginalis, a freshwater edible Mollusca and still underexploited for its health benefit molecules. The low molecular weight ACE inhibitory peptide has been evaluated for enzyme kinetics, isothermal titration calorimetry (ITC), and site-specific docking simulation study. Lineweaver–Burk plot analysis and ITC reveal that peptide-IGPGPFSR is a non-competitive inhibitor. Molecular docking simulation further confirms that this antihypertensive peptide bound to ACE by hydrogen bond interaction between Asp410, Lys468, His470, Arg479, and Glu104 of the enzyme with Ile1, Pro3, Gly4, Ser7, and Arg8 of the peptide respectively. Interaction of Pro5 and Phe6 of the peptide displaces the zinc (Zn²⁺) ion from the active site which results in the incapability of the substrate: Hippuryl-L-Histidyl-L-Leucine (HHL) to bind to the active site of the enzyme and ultimately leads to inhibition of ACE. Present observations suggest that this novel antihypertensive octapeptide-IGPGPFSR is a potent inhibitor of ACE and can be used as an auxiliary for nutraceutical therapy against hypertension. Moreover, the underlying mechanism of ACE inhibition will help in widening our knowledge of drug design against enzyme targets. Graphical Abstract
The Ramachandran plot and the ProSA-web server were used to validate the tertiary structure of the model protein. a Ramachandran plot shows that the number of amino acid residues in the favorable region is 92.59%. b The ProSA-web result gives a Z score of − 7.78.
Identified conformational B-cell epitopes by SEPPA 3.0. a 3D representation of the predicted discontinuous residues on envelope protein was shown as balls. Color of balls was red when score > = 0.2 and salmon when score > = 0.17. b Predicted residues.
Identified conformational B-cell epitopes by ElliPro server. a 3D representation of the predicted discontinuous residues on envelope protein was shown as a spacefill model with a score 0.962. b Predicted residues have a score 0.962 by ElliPro. c 3D representation of the predicted discontinuous residues on envelope protein was shown as a spacefill model having score of 0.936. d Predicted residues having a score 0.936 by ElliPro server.
The consensus predicted conformational B-cell epitopes and residues involved in discontinuous epitope formation on envelope protein by both SEPPA 3.0 and Ellipro tools.
The current spread of Zika virus infection in India has become a public health issue due to the virus’s possible link to birth abnormalities and neurological disorders. There is a need for enhanced vaccines or drugs as a result of its epidemic outbreak and the lack of potential medication. B-cell mediated adaptive immunity is capable of developing pathogen-specific memory that confers immunological protection. Therefore, in this study, the envelope protein of the Zika virus was retrieved from the NCBI protein database. The ABCpred and BepiPred software were used to discover linear B-cell epitopes on envelope protein. Conformational B-cell epitopes on envelope protein were identified using SEPPA 3.0 and Ellipro tools. Predicted B-cell epitopes were evaluated for allergenicity, toxicity, and antigenicity. Two consensus linear B-cell epitopes, envelope165−180 (AKVEITPNSPRAEATL) and envelope224−238 (PWHAGADTGTPHWNN) were identified using ABCpred and BepiPredtools. SEPPA 3.0 and Elliprotools predicted consensus conformational envelope98−110 (DRGWGNGCGLFGK) and envelope248−251 (AHAK) epitopes and one residue (75PRO) within envelope protein as a component of B-cell epitopes. These predicted linear and conformational B-cell epitopes will help in designing peptide vaccines that will activate the humoral response. However, in-vitro and in-vivo laboratory experimental confirmations are still needed to prove the application’s feasibility.
Histopathological lam in the studied groups. Negative control group (H), positive control group (A), gold nanoparticle receiving group (G), peptide receiving group (P), peptide-nanoparticle receiving group (GP)
Changes in IL-4, IL-5, and IL-13 levels in the study groups. Negative control group (H), positive control group (A), gold nanoparticle receiving group (G), peptide receiving group (P), peptide-nanoparticle receiving group (GP)
Gene expression of IL-4, IL-5, IL-13, IL-33, INF-γ, Muc5a and Cox2 in the study groups. Negative control group (H), positive control group (A), gold nanoparticle receiving group (G), peptide receiving group (P), peptide-nanoparticle receiving group (GP)
Asthma is an inflammatory disease that affects the airways. Inflammation of the airway and reversible bronchoconstriction eventually lead to obstruction of the airways, breathlessness and asthma attack. Gold nanoparticles are among the most widely used nanoparticles and have many applications for pharmaceutical and medical purposes. Cortistatin is a neuropeptide that exhibits anti-inflammatory effects. In this study, spherical gold nanoparticles were conjugated to the Cortistatin peptide, for effective and targeted drug delivery to the airways of asthmatic mice. After synthesizing spherical gold nanoparticles and conjugation the Cortistatin, asthma model was produced and treatment with a nano-drug. The levels of inflammatory cytokines, Specific and total IgE, inflammatory gene expression, the number of goblet cells, and the amount of mucus secretion and eosinophilic inflammation in lung tissues were studied. The results showed that Cortistatin-nanoparticle could control allergic and inflammatory factors and the anti-inflammatory effects of Cortistatin peptide-bound nanoparticle are far greater than those of the peptide alone. Increasing the anti-inflammatory effect of peptide-bound nanoparticle versus peptide alone, confirmed the effective, targeted and correct drug delivery of peptide-nanoparticle combination to inflamed areas in lung tissue.
Lactic acid bacteria (LAB) are economically important because of their fermentative aptitude and they also confer health and nutritional benefits. Pediococcus acidilactici NCDC 252 (NCDC 252) is potential probiotic with novel genome that exhibited all in-vitro probiotic attributes, colonization in intestine and survival in conditions that mimick gastric, intestinal and vaginal fluid. Genome was screened for its ability to produce antimicrobial peptide (AMP). AMP from NCDC 252 was isolated and purified against indicator strain (Escherichia coli). AMP production was optimized in MRS broth at 32 °C, pH 6 and upto 36 h of incubation under both aerobic and anaerobic conditions. AMP was purified 2.2 fold with yield of 7.2% and specific activity of 64.5 U mg/mL using ammonium sulphate precipitation (upto 70%) and gel filtration chromatography. Molecular weight of purified AMP was calculated to be 2.5 KDa. Tricine SDS-PAGE and LC/MS studies confirmed it to be a low molecular weight peptide. AMP was stable over pH range of 3 to 9 and temperature range of 40 °C to 100 °C. Enzymatic treatment inactivated the AMP whereas, detergent treatment enhanced its activity. Biopreservation potential of purified AMP and whole NCDC 252 cells was studied by incubating them with some dairy products, apple juice, apricot pulp and pre-pasteurized wine over 72 h. Bacterial growth in AMP treated food products reduced significantly after 72 h. Viable cell count decreased to approximately half in both purified AMP treated and NCDC 252 treated food products. Purified AMP demonstrated exceptional resistance to heat treatment over a wide pH range, enabling it to establish itself as a biopreservative. Studies on biopreservation potential revealed NCDC 252 derived AMP to be effective even at low concentrations and it can be utilised to extend the shelf life of food goods.
(a) Sephadex G-50 chromatography of Naja naja oxiana venom read by spectrophotometer. (b) A view of electrophoresis gel related to fractions and crude venom of Naja naja oxiana
(a) Viability of U-87MG cells treated with partially purified cytotoxic peptides of Naja naja oxiana venom (PPCPNnov) at the concentrations of 0–40 µg/ml after 24 h. (b) Morphological changes in U-87MG cells treated with PPCPNnov at the concentrations of 10, 13.5, and 17 µg/ml after 24 h (Scale bar, 250 μm). The results are expressed as the mean ± SD from three independent experiments, ***p < 0.001
(a) Fluorescence microscopy images and (b) fluorimetry analysis of ROS generation in U-87MG cells after 24 h exposure to different concentration of partially purified cytotoxic peptides of Naja naja oxiana venom (PPCPNnov; 10, 13.5 and 17 µg/ml). The results are expressed as the mean ± SD from three independent experiments, *p < 0.05, ***p < 0.001. Scale bar, 250 μm
(a) Flow cytograms of Annexin V-FITC/PI double stained U-87MG cells exposed to partially purified cytotoxic peptides of Naja naja oxiana venom (PPCPNnov) at the concentration of 10, 13.5 and 17 µg/ml for 24 h. (b) Flow cytometry analysis of U-87MG cells showing percentage of the viable cells (Q3, Annexin V-/PI-), the cells at the early apoptosis (Q1, Annexin V+/PI-), the cells at the late apoptosis (Q2, Annexin V+/PI+), and the necrotic cells (Q4, Annexin V-/PI+). Data expressed as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01 ***p < 0.001
(a) Representative ethidium bromide-stained gel electrophoresis of cDNA amplified with real-time PCR (b) BAX/BCL-2 mRNA expression ratio genes in U87-MG cells treated exposed to partially purified cytotoxic peptides of Naja naja oxiana venom (PPCPNnov) at the concentration of 10, 13.5 and 17 µg/ml for 24 h. The mRNA expression levels are displayed as fold change compared to the β-actin as housekeeping gene. The results are expressed as the mean ± SD from three independent experiments, **p < 0.01
The aim of this study is to assess the cytotoxic effects of partially purified cytotoxic peptides of Naja naja oxiana venom (PPCPNnov) in human Glioblastoma cell line (U-87MG cells). PPCPNnov were prepared by gel filtration of crude venom. U-87MG cells were exposed to different concentrations of PPCPNnov (10, 13/5 and 17 µg/ml). Results showed that PPCPNnov dose-dependently increased the reactive oxygen species (ROS) generation and the percentage of apoptotic cells. Apoptosis at the concentration of 17 µg/ml was accompanied by an increase in the ratio of mRNA expression of BAX/BCL-2. It seems the production of hydrogen peroxide (H2O2) during the enzymatic reaction causing activation of caspase and/or the interaction of PPCPNnov with membrane receptors leading to the cell death process that should be further evaluated.
Alzheimer’s disease (AD) a key health problem chiefly in industrialized countries is characterize by memory loss in older persons. Treatments for AD include psychotropic mediators, disease-modifying managements, psychosocial interventions, and especially inhibition of cholinesterase (AChE) enzyme that will block the hydrolysis of a neurotransmitter acetylcholine (ACh). In the current study, it is reported for the first time that Anisomycine and Puromycin inhibit the activity of the AChE enzyme of krait venom. The results revealed that both Anisomycine (0.8–2.4 mM) and Puromycin (36.8–73.6 µM) exhibited AChE in a dose-dependent procedure. A kinetic study using the plot of Lineweaver Burk showed that both Anisomycine and Puromycin caused a competitive type of inhibition against AChE. The antibiotics (Anisomycin and Puromycin) compete with ACh for binding to the enzyme AChE active site. The Km of venom AChE for Anisomycin (0.8–1.6 mM) increased from 64 to 161% and the Vmax remains constant while for Puromycin (36.8–73.6 µM) the Km increased from 34 to 180% and did not affect the Vmax. The calculated IC50 for Anisomycine was found to be 1.4 mM while for Puromycine it was found to be 36.5 µM. In comparison, Puromycine has a more potent inhibitory effect than Anisomycine. It is concluded, that Anisomycin and Puromycin may possess the anti-AChE ability against Krait snake venom acetylcholinesterase.
Human respiratory syncytial virus (RSV) has been involved in human pediatric lower respiratory tract infections. The intermolecular interaction between RSV phosphoprotein (P) and nucleoprotein (N) plays a vital role in formation of the viral RNA polymerase, which is formed as a peptide-mediated interaction by binding the flexible C-terminal tail of P protein to the rigid globular domain of N protein. In this study, it is revealed that the free P’s C-terminal tail is intrinsically disordered in unbound state, but would fold into a well-structured helical conformation when binding to N protein, thus characterized by a so-called coupled folding-upon-binding process. The Phe241 residue at the end of P’s C-terminus is responsible for the recognition and binding of P protein to N protein, which serves as an anchor residue to root in the binding pocket of N protein and confers both stability and specificity to the N–P interaction. The C-terminal tail’s peptide segment of 9 amino acids long represents a P-peptide; its binding potency to N protein can be regarded as a compromise between favorable direct readout and unfavorable indirect readout. Here, we rationally designed molecular stapling to constrain the free P-peptide into a native-like conformation in unbound state, thus largely minimizing the unfavorable indirect readout effect upon its binding to N protein. Further biophysical characterizations substantiated that the binding affinities of two stapled P-peptide counterparts, namely hs[234,238]P-peptide and hs[235,239]P-peptide, were improved considerably upon the stapling. The stapled peptides may be further exploited as potent self-inhibitory agents to target and disrupt the coupling event of P protein recognition by N protein as anti-RSV therapeutic strategy.
The white spot disease causes significant damage to global aquaculture production. A prominent vaccine, eliciting the immunogenicity of freshwater fishes against Ichthyophthirius multifiliis yet to be developed. Thus, an Immunoinformatic drive was implemented to find out the potential epitopes from the surface immobilization antigens. B-cell derived T-cell epitopes are promiscuous elements for new generation peptide-based vaccine designing. A total of eight common B and T-cell epitopes had filtered out with no overlapping manner. Subsequently, the common epitopes are linked up with EAAAKEAAAKEAAAK linker peptides, we also added L7/L12 ribosomal protein adjuvant at the N- terminal side of peptide sequence for eliciting the immune response in a better way. The secondary and tertiary structural properties of the modeled 3D protein revealed that the protein had all the properties required for a protective immunogen. Afterward, three globally used validation server: PROCKECK, ProSA and ERRAT were used to justify the proper coordinate. NMR, Crystallographic range and error plot calculation for vaccine model also been done respectively. This was followed by molecular docking, MD simulation, NMA analysis, in silico cloning and vaccine dose-based immune response simulation to evaluate the immunogenic potency of the vaccine construct. The in silico immune simulation in response to multi-epitopes show antibody generation and elevated levels of cell-mediated immunity during repeated exposure of the vaccine. The favourable results of the in silico analysis significantly specify that the vaccine construct is really a powerful vaccine candidate and ready to proceed to the next steps of experimental validation and efficacy studies. Graphical Abstract
Antimicrobial peptides (AMPs) have been found to be effective in neutralizing lipopolysaccharide (LPS) due to their cationic characteristics. LHH1 and LGH2 are two AMPs that we previously identified from the genome of Lactobacillus casei HZ1, which is originated from Chinese traditional fermented milk. In present study, the anti-inflammatory capacity of the two AMPs was investigated via a LPS-induced RAW264.7 macrophages inflammation model. Cell viability assay results showed that the AMPs showed a lower cytotoxicity on RAW264.7 cells at a concentration of lower than 32 μM. Compared to LGH2, LHH1 exhibited a good anti-inflammatory effect with a markedly declined release of nitric oxide (NO) from RAW264.7 cells. What’s more, LHH1 could effectively decrease the secretion of pro-inflammatory TNF-α, IL-6 and IL-1β. ITC data depicted that LHH1 could interact with LPS in an exothermic reaction. These data confirmed the efficacy of LHH1 from Lactobacillus casei HZ1 in exerting anti-inflammatory activity, which provided a novel AMP candidate in treating inflammation-related diseases.
Effect of EWP (50, 100, 500 and 1000 μg/mL) on the cell viability, phagocytic activity, NO and cytokines expression of RAW264.7 cells. a Cell viability, b phagocytic activity, c NO, d IL-6, e IL-10, f TNF-α. The data are expressed as the means ± standard deviations (n = 10). Samples with different lowercase letters were significantly different (p < 0.05)
Body weights and organs index of mice; Numbers of white blood cells and contents of hemoglobin in peripheral blood. a Body weights, b organs index, c white blood cells, d hemoglobin. Samples with different lowercase letters were significantly different (p < 0.05)
Effect of EWP on the spleen histological structure in mice
Effect of EWP on the levels of cytokines in spleen tissues. a IL-2, b IL-6, c IL-10, d TNF-α. Samples with different lowercase letters were significantly different (p < 0.05)
Effect of EWP on the level of SOD, T-AOC and MDA in spleen tissues. a SOD, b T-AOC, c MDA. Samples with different lowercase letters were significantly different (p < 0.05)
In this study, the immunomodulatory function of egg white peptides (EWP) was evaluated using RAW264.7 macrophage cells and an immunosuppressive BALB/c mice model. In vitro, 100 μg/mL EWP significantly increased cell viability and upregulated expression of immunomodulators (NO, IL-6, IL-10 and TNF-α) in RAW264.7 cells, which contributed to macrophage activation. In vivo, 150 mg/kg/day EWP alleviated splenomegaly and rebound thymic hyperplasia in BALB/c mice during recovery from immunosuppression. Numbers of white blood cells in peripheral blood and levels of cytokines (IL-2, IL-6, IL-10 and TNF-α) in spleen tissue were significantly increased in mice given 150 mg/kg/day EWP. Spleen pathological changes and oxidation degree showed that EWP promoted the recovery of spleen tissue from immunosuppression and oxidation. Oligopeptides with common characteristics of immunomodulatory peptides might be responsible for the immunomodulatory function of EWP. Therefore, EWP may be considered as a natural immunomodulator that can help immune function recover from immunosuppressive state.
Smp43 is a novel cationic antimicrobial peptide (AMP) which was extracted from the venom of Scorpio maurus palmatus scorpion. However, many studies described the cytotoxic activities of Smp43 on various cancer cell lines; cytotoxicity and its mode of action on human breast cancer remain unstudied. The purpose of this research is to determine the cytotoxicity and the molecular mechanisms of Smp43 in human breast cancer cell lines (MDA-MB-231 and MCF-7). Cells were treated with Smp43 and various assays have been performed including MTT assay, apoptosis assay (Annexin V/PI staining), cell cycle analysis, DNA fragmentation by DPA and agarose gel electrophoresis, and wound healing assay were performed. In addition, apoptosis-related gene expression levels were determined by qRT-PCR while the expression levels of cell proliferation/migration/invasion-related genes were determined by western blotting. Treatment with Smp43 inhibited cell proliferation, migration, and metastasis, but it induced cell apoptosis as observed by DNA fragmentation and Annexin V/PI analysis. Further molecular mechanism studies showed that bax, p53, caspase 7, and caspase 9 expression levels was found to be up regulated in both treated cell lines. On the other hand, bcl-2, ki67, PCNA, laminin-5, and upA expression levels significantly downregulated in both treated cell lines. These findings were also validated by ELISA test of cytochrome C, MMP9, and VEGF. Generally, our results revealed that proliferation of breast cancer cells is dramatically reduced in vitro by Smp43 through apoptosis induction and migration/invasion inhibition. Our findings provide new insights about antitumor activity of scorpion venom antimicrobial peptides and may lead to the development of effective therapeutic agents targeting breast cancer.
In the design and development of therapeutic agents, macromolecules with restricted structures have stronger competitive edges than linear biological entities since cyclization can overcome the limitations of linear structures. The common issues of linear peptides include susceptibility to degradation of the peptidase enzyme, off-target effects, and necessity of routine dosing, leading to instability and ineffectiveness. The unique conformational constraint of cyclic peptides provides a larger surface area to interact with the target at the same time, improving the membrane permeability and in vivo stability compared to their linear counterparts. Currently, cyclic peptides have been reported to possess various activities, such as antifungal, antiviral and antimicrobial activities. To date, there is emerging interest in cyclic peptide therapeutics, and increasing numbers of clinically approved cyclic peptide drugs are available on the market. In this review, the medical significance of cyclic peptides in the defence against viral infections will be highlighted. Except for chikungunya virus, which lacks specific antiviral treatment, all the viral diseases targeted in this review are those with effective treatments yet with certain limitations to date. Thus, strategies and approaches to optimise the antiviral effect of cyclic peptides will be discussed along with their respective outcomes. Apart from isolated naturally occurring cyclic peptides, chemically synthesized or modified cyclic peptides with antiviral activities targeting coronavirus, herpes simplex viruses, human immunodeficiency virus, Ebola virus, influenza virus, dengue virus, five main hepatitis viruses, termed as type A, B, C, D and E and chikungunya virus will be reviewed herein. Graphical Abstract
Secondary structure of peptide BcI-1003, performed by Pepfold-3 server and visualized with Discovery Studio Software (Biovia)
2D representation of the complex peptide-enzyme interaction (Discovery Studio Visualizer 2021-Biovia). Left: complex BcI-1003/BChE, right: complex BcI-1003/MAO-B
DC spectra of BcI-1003 obtained in the following experimental conditions: Milli Q water, TFE/H2O (50% v/v) and in the presence of DPPG and (DPPC) vesicles
Nature continues to be one of the most important sources of molecules for the development of novel therapeutic agents. The skin of anuran’s (frogs and toads) is a rich source of peptides with a great importance in the search of bioactive agents applying to human health. Alzheimer's disease (AD) is a complex disease associated with numerous pathological pathways, making their simultaneous modulation necessary. On the other hand, the increasing bacterial resistance against conventional antibiotics has made it essential to search for new antimicrobial drugs with different modes of action. Here in we report the natural peptide BcI-1003, isolated from Boana cordobae amphibian skin, as an agent capable to act on three key therapeutic targets of AD, inhibiting the activity of BChE (IC50 = 669 µM) and MAO-B (IC50 = 570 µM) enzymes, and showing a powerful and rapid antioxidant activity (EC50 = 7.24 µM). Besides, BcI-1003 showed antimicrobial activity against clinically drug-resistant gram-positive and gram-negative bacterial strains, with MIC values ranging from 8 to 127 µM against Staphylococcus aureus MR-1; S. aureus MR-2 and Escherichia coli MDR-1.
In spite of existing cases of severe viral infections with a high mortality rate, there are not enough antiviral drugs and vaccines available for the prevention and treatment of such diseases. In addition, the increasing reports of the emergence of viral epidemics highlight, the need for novel molecules with antiviral potential. Antimicrobial peptides (AMPs) with antiviral activity or antiviral peptides (AVPs) have turned into a research hotspot and already show tremendous potential to become pharmaceutically available antiviral medicines. AMPs, a diverse group of bioactive peptides act as a part of our first line of defense against pathogen inactivation. Although most of the currently reported AMPs are either antibacterial or antifungal peptides, the number of antiviral peptides is gradually increasing. Some of the AMPs that are shown as effective antivirals have been deployed against viruses such as influenza A virus, severe acute respiratory syndrome coronavirus (SARS-CoV), HIV, HSV, West Nile Virus (WNV), and other viruses. This review offers an overview of AVPs that have been approved within the past few years and will set out a few of the most essential patents and their usage within the context mentioned above during 2000–2020. Moreover, the present study will explain some of the progress in antiviral drugs based on peptides and peptide-related antivirals.
Charts of TG (the solid line) and DTG (the dotted line) from Pep (A), Pap (B), Nep (C), Prp (D), Trp (E), Alp (F), Snp (G) and their FTIR spectra (H), respective
The degree of hydrolysis (DH) from PEP (A), PAP (B), NEU (C), PRO (D), TRY (E), ALK (F) and SNA (G) methods. Each value is the mean ± SD of triplicate measurements (n = 3)
Amimo acid content (A) and increment (B) of seven enzymatic polypeptides, including Pep, Pap, Nep, Prp, Trp, Alp and Snp, respective. Asp aspartic acid, Thr threonine, Ser serine, Glu glutamic acid, Gly glycine, Ala alanine, Cys cysteine, Val valine, Met methionine, Ile isoleucine, Leu leucine, Tyr tyrosine, Phe phenylalanine, His histidine, Lys lysine, Trp tryptophan, Arg arginine and Pro proline. Each value is the mean ± SD of triplicate measurements (n = 3)
Antioxidant activities of seven enzymatic polypeptide on lipid peroxidation (A), superoxide anion (B), DPPH radical (C), hydroxyl radical (D), ABTS radical (E). Vc Vitamin C, Pep pepsin enzymatic polypeptide, Pap papain enzymatic polypeptide, Nep neutral protease enzymatic polypeptide, Prp protemax enzymatic polypeptide, Trp trypsase enzymatic polypeptide, Alp alkaline protease enzymatic polypeptide, Snp snail protease enzymatic polypeptide and Raw raw material. Each value is the mean ± SD of triplicate measurements (n = 3)
The α-glucosidase inhibition activity (A) and α-amylase (B) inhibition activity of Pep, Pap, Nep, Prp, Trp, Alp and Snp. Aca Acarbose, Pep pepsin enzymatic polypeptide, Pap papain enzymatic polypeptide, Nep neutral protease enzymatic polypeptide, Prp protemax enzymatic polypeptide, Trp trypsase enzymatic polypeptide, Alp alkaline protease enzymatic polypeptide, Snp snail protease enzymatic polypeptide and Raw raw material. Each value is the mean ± SD of triplicate measurements (n = 3)
As a new food raw material, Acer truncatum seed has been used in the production of edible oil. However, the research on the processing residue (seed meal) has not been reported. The Acer truncatum seed meal was hydrolyzed by seven enzymes and the enzymic polypeptides obtained were characterized by thermogravimetric analysis, gel permeation chromatography, fourier transform infrared spectroscopy and zeta pals dynamic light scattering. In addition, the degree of hydrolysis, amino acid composition, antioxidant activity, α-glucosidase inhibition activity and α-amylase inhibition activity of enzymatic polypeptide were evaluated. The results showed that the yields, average molecular weights, average particle sizes and zeta potentials of enzymic polypeptides obtained were 20.5–40.2%, 7.8–13.9 kDa, 288.3–997.5 nm and ( −)1.8 to ( −)36.4 mV, respectively. The bioactivity results showed that the enzymatic polypeptides not only had higher antioxidant activity, but also better α-glucosidase and α-amylase inhibition activities than the raw materials. Among the seven enzymatic polypeptides, the alkaline protease enzymatic polypeptide had the highest content of the eight essential amino acids (20.3%), the largest value of hydrolysis degree (15.4%) and the most significant bioactivity. The study will provide the data support for developing the new application of seed meal from Acer truncatum.
The subgenus Tityus (Archaeotityus) includes small variegated species considered until recently, a basal group within Tityus, one of the most diverse and medically relevant genera of scorpions in the New World. Archaeotityus species are distributed in the humid forests of Lower Central America and the Choco, Guiana Shield, and Amazonian regions. Due to their size and habits, Archaeotityus species are not usually considered dangerous, however, there are some clinical reports that show otherwise. To contribute to the toxinological knowledge of these poorly explored species, we characterized the venom of Tityus ocelote from three localities in Costa Rica. In addition, we assessed the evolutionary relationships of putative sodium channel-modulating peptides found in this species with those reported for other members of the genus, through a maximum likelihood phylogenetic analysis based on their amino acid sequences. We observed the presence of homologs of previously identified NaTx from the species Tityus (Archaeotityus) clathratus and some other putative Na+ and K+ channel modulating peptides related to the T. bahiensis group. In addition, we sequenced some peptides related to toxins present in the venom of the subgenus Atreus, such as those reported for T. obscurus, T. pachyurus, and the Costa Rican endemic species T. dedoslargos. Our phylogenetic analysis suggests that the venom of this Archaeotityus species is very complex and that some of the ion channel toxins expressed in it are related to distinct lineages within the genus Tityus, which could represent a plesiomorphic condition conserved in this group of scorpions of the New World.
The pathogenesis of hypertension is related to many factors including active angiotensin-I-converting enzyme (ACE) and excess reactive oxygen species (ROS), the combination of ACE inhibition and antioxidant activity in one compound might be useful for the treatment of hypertension. Sipunculus nudus L contain high protein content, which can be used as a valuable raw material to prepare bio-active peptides. In this study, two novel tripeptides, LPK and PRP, with dual bioactivity of ACE inhibition and antioxidant were purified and identified from the worm. The antioxidant activities of LPK and PRP are dose-dependent, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion and hydroxyl radical scavenging activities. Moreover, the antioxidant activity of PRP was stronger than that of LPK. In addition, LPK and PRP also show higher ACE inhibitory activity, with IC50 of 6.54 and 0.42 mM, respectively. Molecular docking simulation showed that both the peptides could interact with the active ACE sites via hydrogen bonds, which can reduce the production of Ang II, thereby; LPK and PRP treatment could decrease Ang II-triggered superoxide production, and meanwhile, the peptides treatment could directly remove excess reactive oxygen species. The above-mentioned advantageous effects appeared to involve the stress of the RAS and scavenging ROS, both were benefit to treat hypertension. This research showed that LPK and PRP had potential to be developed as antihypertension agents, which might be a new strategy for high value utilization of Sipunculus nudus L as functional food material.
CuSO4 was used and created an in-vivo inflammatory model of a zebrafish embryo for drug testing in this work. In our study, molecular docking was performed to check the possible polar interaction with the peptide, TL15 of sulphite reductase derived from spirulina (Arthrospira plantensis) and the COX-2, which is predicted to have anti-inflammatory activity. In-vitro anti-inflammatory activity was performed on human erythrocytes, and the potential activity of TL15 peptide was in a concentration-dependent manner. We further investigated the technique for evaluating the anti-inflammatory effects of TL15. To produce acute inflammation, the zebrafish (Danio rerio) larvae were treated with CuSO4 at 2 μM and toxicity analysis were done at 0–96 hpf. The effect of TL15 was analysed upon treatment to CuSO4 induced larvae at dose-dependent manner (10–80 μM) and exhibited a gradual reduction in oxidative stress at 80 μM TL15. Macrophages were monitored using neutral red labelling after CuSO4 stimulation; as the major end-point, larval mortality was employed. RT-PCR was used to assess the expression level of important cytokines implicated in the inflammatory response, such as COX-2, TNF-α, IL-1β and IL-10. Within 24 h, CuSO4 increased mortality in a dose-dependent process. The increment in the inflammatory response due to CuSO4 induction in zebrafish larvae was demonstrated by analysing the COX-2, IL-1β, IL-10 and TNF-α expression. In addition, we also investigated the reactive oxygen species scavenging activity of TL15 on CuSO4 induced larvae by analysing the GST and GPX expression levels in the zebrafish embryo. Overall, TL15 of sulphite reductase was found to have a potent anti-inflammatory activity by scavenging the free radical productions.
Non-small-cell lung cancer (NSCLC) is the most common lung cancer which has the highest mortality rate in Indonesia. One of the trends in treating cancer is by utilizing peptide vaccines, an immunotherapeutic approach that aims to stimulate the cell-mediated adaptive immune system to recognize cancer-associated peptides. Currently, no peptide vaccines are available in the market for NSCLC treatment. Therefore, this project aims to develop a multi-epitope peptide-based vaccine for NSCLC utilizing citrullinated peptides. Citrullination is a post-translational modification that occurs in cancer cells during autophagy that functions to induce immune responses towards modified self-epitopes such as tumor cells, through activation of PAD enzymes within the APC and target cells. It was found that introducing a common citrullinated neo-antigen peptide such as vimentin and enolase to the immune system could stimulate a higher specific CD4⁺ T cell response against NSCLC. Moreover, carcinoembryonic antigen (CEA), an antigen that is highly expressed in cancer cells, is also added to increase the vaccine’s specificity and to mobilize both CD4⁺ and CD8⁺ T cells. These antigens bind strongly to the MHC Class II alleles such as HLA-DRB1*07:01 and HLA-DRB*11:01, which are predominant alleles in Indonesian populations. Through in silico approach, the peptides generated from CEA, citrullinated vimentin and enolase, were analyzed for their MHC binding strength, immunogenicity, ability to induce IFNγ response, and population coverage. It is expected that the immunodominant antigens presentation is able to induce a potent immune response in NSCLC patients in Indonesia, resulting in tumor eradication.
Antagonistic activities of B. safensis strain MK-12.1 against indicator strains
Optimization of incubation time and temperature for growth and bioactive compound production by B.safensis MK-12.1
Culturing medium optimization for B. safensis MK-12.1 growth and bioactive compounds production. Trypticase soy broth: TSB, Nutrients broth: NB, Luria Bertani LB, Brain heart infusion: BHI and Optimized: OPT
Optimization of pH for growth and bioactive compounds production by B.safensis MK-12.1
Evolutionary analysis of B. safensis strain MK-12.1 with related Bacillus species. Phylogenetic tree was generated based 16S rRNA with neighbor-joining method using MEGA X
Antimicrobial resistance in pathogenic bacteria is increasing rapidly. Therefore, investigation of novel therapeutic agents are essential to combat the growing multidrug resistance (MDR) in human pathogens. The current study was intended to identify antibacterial metabolites producing Bacillus spp. from the unexplored southern region of Pakistan. The bacterial isolates (n = 47) were isolated from soil samples and screened for antibacterial activity against a set of American Type Culture Collection (ATCC) and MDR human pathogenic bacterial strains. Among all isolated Bacillus spp. the strain MK-12.1 exhibt promising antibacterial activities agains all tested strains. The antibacterial metabolites from MK-12.1 were partially extracted and their molecular weight and toxicity were evaluated. Genomic DNA was extracted from a fresh culture of antibacterial metabolite-producing strain MK-12.1, and the 16S rRNA gene was amplified and sequenced. The growth and antibacterial metabolites production by MK-12.1 was optimized at various pH, temperature, incubation time, and medium. The strain MK-12.1 efficiently inhibit the growth of ATCC as well as MDR pathogenic bacterial strains with the zone of inhibition ranges from 14 to 18 mm. The partially extracted antibacterial compound was characterized and confirmed to be proteinogenic, non-hemolytic, and molecular weight was less than 20 kDa. The isolate MK-12.1 was identified as Bacillus safensis and revealed 99% similarity with B. safensis strain NBRC. Maximum antagonistic activity and growth of MK-12.1 were observed at pH 8, 30 °C temperature, and 48 h of incubation in a shaking incubator when fermented in an optimized (opt) medium. The current study results indicate that B. safensis MK-12.1 produces promising antibacterial compounds that could be a source of new antibacterial leads to fight against MDR bacteria in future.
Schematic workflow of the common hypothetical proteins in silico. Common hypothetical proteins of resistant strains were obtained and then structural, functional and immunological predictions were made for these proteins. Finally, two proteins were introduced as vaccine and drug candidates
Models of third structure of 10 hypothetical proteins predicted by the I-TASSER server, a HP1, b HP2, c HP3, d HP4, e HP5, f HP6, g HP7, h HP8, i HP9, and j HP10
Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, characterized by increasing antibiotic resistance, severity and mortality. Therefore, numerous efforts have been made nowadays to identify new therapeutic targets. This study aimed to find potential drug targets and vaccine candidates in drug-resistant strains of P. aeruginosa. Extensive antibiotic-resistant and carbapenem-resistant strains of P. aeruginosa with complete genome were selected and ten common hypothetical proteins (HPs) containing more than 200 amino acids were obtained. The structural, functional and immunological predictions of these HPs were performed with the utility of bioinformatics approaches. Two common HPs (Gene ID: 2877781645 and 2877781936) among other investigated proteins were revealed as potential candidates for pharmaceutical and vaccine purposes based on structural and physicochemical properties, functional domains, subcellular localizations, signal peptides, toxicity, virulence factor, antigenicity, allergenicity and immunoinformatic predictions. The consequence of this predictive study will assist in novel drug and vaccine design through experimental investigations.
Lunasin is a soybean peptide with promising therapeutic applications in cancer and other diseases. It is described as an intrinsically disordered peptide, monomeric, and that can form an intramolecular disulfide bond between Cys10 and Cys22. In this study, we tested a new approach to obtain recombinant lunasin by Escherichia coli expression and performed its structural characterization. We expressed a lunasin sequence with an N-terminal 6× His-tag, B1 domain of Streptococcal protein G (GB1), and Tobacco etch virus (TEV) cleavage site (His6-GB1-lunasin), using pET-25b(+) vector. His6-GB1-lunasin purification using immobilized metal affinity chromatography achieved high recovery. We obtained a final yield of 12.0 (± 0.39) mg/L of recombinant lunasin with high purity. The molecular mass and conformation were as expected, as verified by liquid chromatography–mass spectrometry (LC–MS), electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI–IMS–MS), electrospray ionization–mass spectrometry (ESI–MS), size-exclusion chromatography, circular dichroism, fluorescence spectroscopy, and one-dimensional ¹H nuclear magnetic resonance. Additionally, the identity of lunasin and its complete amino acid sequence was confirmed by LC–MS/MS. Recombinant lunasin also showed antioxidant activity (2.92 ± 0.16 µmol of trolox equivalents/µmol lunasin) by oxygen radical absorbance capacity and inhibited cell migration of MDA-MB-231 cell line. On the other hand, for the first time, LC–MS, ESI–IMS–MS and ESI–MS analyses revealed, in addition to the reduced and oxidized monomeric forms, the presence of small populations of disulfide cross-linked dimeric species of lunasin. Overall, our data demonstrate the effectiveness of the GB1-tagging system to obtain lunasin, that lunasin can form disulfide cross-linked dimers, and that LC–MS, ESI–IMS–MS and ESI–MS are efficient analytical chemistry techniques to assess reduced and oxidized (monomer and dimer) species.
Classification of natural sweetening agent (Priya et al. 2011)
Classification of artificial sweetening agent (Chaudhry and Dutta 2021)
Classification of peptide sweetening agent (Kashani-Amin et al. 2021)
The increasing prevalence of diseases caused by sugar consumption has become a threat to human health, and various studies have reported the relationship between high sugar consumption and the risk of various cardiovascular diseases, obesity, type 2 diabetes. Sugar-free products such as low-calorie sweeteners, especially peptide types, are very popular today due to the production of fewer calories. These sweeteners often have a protein structure and have a wide variety in terms of taste and dosage. Although extensive studies consider sweeteners to be safe and suitable substitutes for sugar, studies show that artificial types of these sweeteners can cause oxidative stress, metabolic syndrome, nervous system diseases, changes in the gastrointestinal microflora. Despite these conflicting studies, food safety organizations such as the FDA, FAO, EFSA limit the consumption of sweeteners to the acceptable daily intake (ADI) for all people, except for cases such as phenylketonuria. The purpose of this study is to briefly introduce natural peptide sweeteners (NPSs) that are good candidates to replace sugar and artificial sweeteners. The most important NPSs discussed in this summary include thaumatin, brazzein, monellin, curculin, miraculin, mabinlin, pentadin, whose safety, dosage and toxicity are discussed. Among the NPSs, thaumatin has been approved by FDA. This protein offers sweetness about 2000 times more than sucrose while produces only 4 kcal/g. NPSs generally show fewer side effects than synthetic types. The use of other NPS is also currently legal as a flavor enhancer and sugar substitute, but there are still challenges to their approval by the FDA.
A Classification of putative allergens into different protein families. The 16 most prominent protein families based on number of allergens present in the particular protein family are plotted. B Gene Ontology (GO term) analysis of putative allergen proteins and the results are plotted on the basis of percentage and number of allergens present in different GO terms
Orthology analysis of putative allergens
Molecular modelling and validation of selected allergen. A Quality check of the template used. B Ramachandra plot generated for the predicted model
A Docked structure with highlighted B-cell epitope of A0A5E4EF81. Rainbow color sites depicts the predicted linear conserved B-cell epitopes. B RMSD of antigen-antibody complex during the molecular simulation analysis for 30 ns
Approximately, 10% of the world population is facing the challenge of food allergy in direct or indirect way. In this study, a genome-wide identification and annotation of the novel putative allergen from almond is performed. Initially, the whole proteome of almond (31,000 proteins) was scanned by Allergenonline, a publically available database of already reported allergens from different sources. The detailed analysis suggests that there are 430 putative allergens which reduced to 45 on motif-based screening using AllFam database. These predicted allergens are annotated for their function by using PFAM, GO databases and orthology analysis. To validate our prediction, we have used structural insights of allergen and antibody interactions for one of the predicted putative allergen protein, homologous to Pru ar 3.0101allergen from apricot. The structure of putative allergen was modeled and molecular docking studies were performed against the antibody. The best docked conformation was subjected to molecular simulation studies to confirm the stable binding of these two molecules. This detailed analysis suggests that the identified allergen will show cross reactivity similar to Pru ar 3.0101 allergen from apricot. This is one of the first report of identifying and annotating the homologous of Pru ar 3.0101 allergen in almond.
Antibody response to Vaxigrip antigen after immunization
* p < 0.05 vs. V group, ⁺ p < 0.05 vs. (V + Alum) group
The comparison of IgG1 and IgG2a antibodies to Vaxigrip at day 42
⁺ p < 0.05 vs. (V + Alum) group
Hemagglutinin inhibition (HI) оf mice sera after immunization with Vaxigrip (V), Stemokin (V + S100 µg), and Alhydrogel (V + Alum) group
Vaccines are widely used worldwide to prevent and protect from various infections. A variety of modern approaches to developing prophylactic and therapeutic vaccines is growing. In almost all cases, adjuvants are necessary to obtain an effective immune response.This work investigated the possibility of using the pharmaceutical peptide drug Stemokin as an adjuvant stimulating a balanced Th1/Th2 response.A study was conducted to compare the activity of Stemokin versus the approved adjuvant Alhydrogel in a murine vaccination model with the approved VAXIGRIP® vaccine.The first proof-of-concept experimental study shows that the peptide Ile-Glu-Trp has the adjuvant vaccine properties and anti-HA IgG2a enhancing response, revealing a Th1- favoring balanced Th1/Th2 immunomodulation.
Sources of Peptides
Biotherapeutic potential mechanism of antioxidant BAPs in the human body
Biotherapeutic potential mechanism of ACE inhibitory BAPs in the human body
Bioactive peptides (BAPs) are natural products which are gaining interest of researcher as many synthetic drugs have shown different side effects while curing various morbidities. BAPs are organic substances formed by 2–20 residues of amino acids joined by covalent bonds. BAPs are considered the new generation of biologically active regulators; they can prevent oxidation and microbial degradation in foods and also improve the treatment of various diseases and disorders, thus increasing the quality of life. This review focus on various sources of BAPs and different production methods to maximize the generation of these important/ubiquitous and useful biomolecules. Moreover, it describes the isolation and fractionation to get pure BAPs, which can exert potential bioactivities on body, including antioxidant, antihypertensive, antimicrobial, immune-modulatory, and anti-cancer effects. On the other hand, it also highlights the role of bioinformatics tools in prediction of bifunctionalities and mode of action of BAPs. Furthermore, it describes various hurdles/challenges related to this field and their possible solutions. This review is expected to cover the significant aspects of bioactive peptides and provide the related information for using these peptides as potential nutraceutical or pharmaceutical ingredients.
Snake venom acetylcholinesterase. A plot of % residual activity and % residual inhibition vs tri-peptides fragment concentration
Inhibition of Krait (Bungarus sindanus) AChE in the presence of various doses of the synthesized tri-peptides fragment. The rates of hydrolysis V were dignified at 412 nm by using a fixed ACh concentration of 0.5 mM (AcSCh) in 1 mL assay solutions with phosphate buffer (62 mM and pH 7.4) and 0.2 mM of DTNB followed by incubation at 37C˚ for 10 min before adding of the substrate. (n = 3), p < 0.05
Krait Venom AChE experiments in the absence and presence of various doses of the tri-peptides fragment as shown in the legend box. All experiments were repetitive at least four times and in all cases, comparable results were acquired
Dipeptide N-Cbz-L-Leu-threo-D, L-Pheser-OH (3) syntheiss
Tri-peptides Z-L-Leu-threo-L- Pheser-L-Phe-OMe (4) and Z-L-Leu-threo-D-Pheser-L-Phe-OMe (5) synthesis
In the current study, the cyclopeptide alkaloid discarine D-derived tri-peptides fragments were synthesized and then investigated for their inhibitory potential against krait (Bungarus sindanus) venom acetylcholinesterase (AChE) enzyme. The tri-peptides L-Leu- threo-D-Pheser-L-Phe and L-Leu- threo-L-Pheser-L-Phe were chemically synthesized by a conventional method using the benzyloxycarbonyl group for the alpha-amino (α-amino) safety and the methyl esters an amino acids derivative used for the safety of carboxyl group. The present paper described that the general synthetic strategy of tri-peptide allows the tri-peptide sequence to be acquired with the N-terminal extreme protected. Kinetic studies using the Lineweaver Burk plot indicated that tri-peptides fragments cause an un-competitive type of inhibition i.e. both Km and Vmax values decreased with the increase of tri-peptides fragment concentration (13.5–22.5 µM). The estimated Ki and IC50 for krait venom AChE were found to be 17.5 µM and 19.5 µM, respectively. Thus the present paper, clarified that the freshly produced tri-peptides fragment can be deliberated as a beneficial mediator for the inhibition of krait venom AChE.
The multi-dimensional applications of shrimp AMPs
Antimicrobial peptides (AMPs) are small peptides playing a lead role in the innate immune system of organisms. Marine organisms have a plethora of AMPs that have been widely explored due to their multitude of functions. This review focuses on shrimp derived AMPs and details their versatile nature from an application perspective. It highlights the applications of shrimp AMPs, such as their role in stress regulation and ontogeny, as alternative sexually transmitted diseases drugs, anticancer agents, immunomodulators, and biomarkers. Ultimately, we are trying to emphasize that shrimp AMPs are beyond mere antimicrobials.
Work architecture used in this study for the construction of the predictive models. For the generation of all predictive models, the scikit-learn and LightGBM libraries were used. Finally, the best predictive models were selected and incorporated into the web app developed with the Flask framework
The user interface of nAChR-PEP-PRED. A Input section of amino acid sequences in FASTA format. Note that the sequences can be entered into the application through the text box or through the load button, which accepts files in.TXT or FASTA format containing the amino acid sequences. B Results section, where the output peptides have an associated probability score that ranges from 1 to 0 for their ranking
Nicotinic acetylcholine receptors (nAChR) are interesting therapeutic targets due to their involvement in the development of different types of diseases. nAChR inhibitory peptides are considered promising drugs due to their high selectivity and activity on these receptors. However, the identification of nAChR inhibitory peptides using conventional in vitro and in vivo assays is time-consuming and expensive. In this sense, machine learning techniques could offer an advantage to deal with these problems. Among machine learning algorithms, the random forest classifier is one of the best performers in classifying peptides with different types of biological activities. Taking into account the aforementioned aspects, in this work we develop a robust bioinformatic tool for the specific prediction of nAChR inhibitory peptides. In this study, three predictive models with good performance measures were generated from the combination of different features selected using the Gini decrease method and the random forest classifier. The best predictive model presented the following performance measures during the fivefold cross-validation on the training data with Accuracy = 0.85, F1-score = 0.87, Precision = 0.85, Specificity = 0.81, Sensitivity = 0.90, Matthew’s correlation coefficient = 0.71; and Accuracy = 0.98, F1-score = 0.98, Precision = 0.95, Specificity = 0.95, Sensitivity = 1.0, Matthew’s correlation coefficient = 0.95 in the testing phase. From the selection of the best predictive model, a bioinformatics tool with a friendly user interface was built, called nAChR-PEP-PRED, which allows the analysis of thousands of amino acid sequences. We believe that this tool can accelerate the discovery of new nAChR inhibitory peptides to reduce the time and costs of conventional experimental assays. Our web tool, nAChR-PEP-PRE, is available at https:// nachr- pep- pred. herok uapp. com/.
Alzheimer's disease (AD) is a common progressive and irreversible neurodegenerative disorder. Neuronal loss in the brain is one of the important characteristic features of AD, which is along with memory and cognitive dysfunction. Activation of programmed cell deaths, especially apoptosis and necroptosis, and autophagy failure play critical roles in the pathogenesis of AD. Ghrelin, an appetite-related hormone, exerts neuroprotective effects via the stimulation of growth hormone secretagogue receptor type 1a in the central nervous system. In this study, rats' cognitive impairments were induced by intra-hippocampal Cornu Ammonis 3 administration of amyloid-beta 1-42 (Aβ 1-42). Then, animals were treated with ghrelin 80 μg/kg via intraperitoneal injection for 10 consecutive days. The Morris water maze and passive avoidance learning test were used to evaluate the effect of ghrelin on learning and memory performance. After that, a histopathological study on rat’s hippocampi was done, and the expressions of pro-apoptotic protein Bax, anti-apoptotic protein Bcl-2, necroptotic proteins RIP1K and RIP3K and autophagic marker Beclin-1 were assessed, using western blotting method. Our findings show that ghrelin improves memory impairment in Aβ 1-42-induced rats and reduces Bax, RIP1K, and RIP3K expressions and also Bax/Bcl-2 ratio, as well as Beclin-1 expressions. In conclusion, our study suggests that ghrelin can provide neuroprotection via inhibition of apoptosis and necroptosis and autophagy promotion in Aβ 1-42-induced model of Alzheimer's disease.
Angiotensin converting enzyme inhibitor is a one of the best anti-hypertension well known drugs for treatment of hypertension (Cushman and Ondetti, Hypertension 17:589–592, 1991). In available ACE inhibitors have various side effects like angioedema, dry cough, skin irritation, and multiple dosing due to less bioavailability (Natesh et al., Nature 421:551–554, 2003, Natesh et al., Biochemistry 43:8718–8724, 2004). We had previously published the data for pharmacological profile and potency of a peptidomimetic compound, 3-Thienylalanine-Ornithine-Proline (TOP) is in-house compound. TOP has shown potent activity and a promising profile in in-vitro, ex-vivo and in-vivo assays as published previously from our laboratory (Seth et al., Drug Des Devel Ther, 2016, Chaudhary et al., J Cardiovasc Pharmacol 61:311–317, 2013). The potent and interesting pharmacological activity shown by this compound further forced us to look at the toxicological profile of this compound in in-vivo in sub-chronic dosing in different dose regime above its pharmacological dose. Therefore, in the current study, we aimed to evaluate the toxicity study that is the 28 days toxicity of our inhouse compound TOP applying the recommended OECD guidelines for safety. In this study, we have taken the dose 10 times higher dose than effective dose. However, our compound (TOP) did not show any toxic sign and symptom and no significantly adverse effect in blood chemistry, hematologic values and histology till higher dose 100 mg/kg. In conclusion, in this study TOP showed promising pharmacological activity without any signs of toxicity in animals.
The effect of Cans on the viability of different cell lines; A the viability of HUVECs was measured by MTT assay, B the viability of HUVECs was tested using trypan blue exclusion dye assay. C and D The effect of Cans on the viability of non-tumorigenic MCF10A, and L929 cell lines measured by MTT assay. Data are presented as mean ± standard error of three independent experiments. Ctrl Control, **P < 0.01, ***P < 0.001 between groups, ****P < 0.0001 vs. control
Evaluation of induction of apoptosis in HUVECs. A and B Annexin V-FITC/PI staining: a; control, b; 20 μM of peptide, c; 30 μM of peptide, and d; 50 μM of peptide. B Data are presented as mean ± standard error of three experiments. **P < 0.01 vs. control, #P < 0.01 vs. treated groups. C Caspase-3 activity was measured using a colorimetric Caspase-3 Assay kit. Data are presented as mean ± standard error of three experiments. **P < 0.01 between treated groups. ns not significant (P > 0.01)
A and B evaluation of expression of Bcl-2 and caspase-8 in HUVECs using RT-qPCR. A Total RNAs were extracted using Trizol reagent and the integrity of the RNA was determined using the 5S, 18S, and 28S rRNA bands following agarose gel electrophoresis. Lane 1: ladder, lane 2: Cans 20 μM, lane 3: Cans 30 μM, and lane 4: Cans 50 μM. B: qPCR analysis of Bcl-2 and Caspase-8 genes was carried out by the RealQ Plus 2 × Master Mix Green without ROX™. GAPDH was used for normalization. Data were analyzed using Relative Expression Software Tool (REST, QIAGEN) and the 2–ΔΔCt method. **P < 0.01 vs. control and #P < 0.01 vs. treated groups. C Immunohistochemical staining of Bax and Bcl-2 proteins in the mouse colon carcinoma sections. The numbers of positive cells were determined by analyzing ten randomly chosen fields and Image J software. D and E The amount of Bax and Bcl-2 proteins was stated as percentages of positive cells per field. **P < 0.01 vs. control
Canstatin, the NC1 domain of the α2 chain of collagen IV, prevents tumor growth through angiogenesis inhibition and apoptosis induction. N-terminal 1–89 amino acid fragment of canstatin induces apoptosis much higher than the C-terminal fragment. Recently, we demonstrated that the amino acids 78–86 of canstatin, so-called Cans, have more anti-migration, anti-tube formation, and anti-tumor activities than other collagen IV derived peptides. Here, we evaluated HUVEC, MCF10A, and L929 cell viability, the percentage of apoptotic cells by Annexin V-FITC/PI staining, caspase-3 activity, Bcl-2, and caspase-8 gene expression using RT-qPCR in endothelial cells, and Bax and Bcl-2 expression in tumors by immunohistochemistry to investigate the apoptotic effect of Cans. Results showed that the peptide reduced the percentage of viable HUVE cells with EC50 of 21 μM and was not toxic for normal cell lines. 30 and 50 μM of Cans induced 34.6% and 50.7% early and late apoptosis in HUVECs compared to 16.5% in control. In addition, caspase-3 activity was amplified up to threefold compared to the untreated cells. Cans down-regulated Bcl-2 and caspase-8 gene expression. This result may show that this peptide acts through the intrinsic pathway and cannot affect the extrinsic pathway of apoptosis. However, this hypothesis requires more investigation. Besides, Bcl-2 reduction and Bax elevation in tumor sections indicated that this peptide could stimulate apoptosis in vivo. In conclusion, we showed that the short canstatin peptide induces apoptosis in endothelial and tumor cells as one of its anti-angiogenic and anti-tumor mechanisms.
Top-cited authors
Subrota Hati
  • Kamdhenu University
Kuo-Chen Chou
  • Gordon Life Science Institute
Amar A. Sakure
  • Anand Agricultural University
Yogesh Gat
  • Lovely Professional University
Vikas Kumar
  • Punjab Agricultural University