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The use of KCF-18, a peptide originating from cytokine receptors, as a therapeutic approach for anti-inflammatory purposes through binding to proinflammatory cytokines.

Cardiovascular diseases such as atherosclerosis, restenosis, and coronary artery disease are linked to chronic inflammation, which current treatment and prevention strategies have proven inadequate to address effectively. A new approach is therefore needed to tackle the problem of inflammation. This study took a novel approach by designing a peptide, KCF18, based on molecular docking and structural analysis.

KCF18 has amphiphilic properties that include positively charged and hydrophobic residues, making it a promising candidate for inhibiting cytokine-induced inflammatory response. The simulations demonstrated that KCF18 could bind to cytokines simultaneously, with electrostatic interactions being the dominant mechanism. Experimental measurements using surface plasmon resonance detection and MM/PBSA binding free energy calculations confirmed that KCF18 was able to bind to both tumor necrosis factor-α (TNF-α) and interleukin-6.

In cell experiments, KCF18 was shown to significantly reduce the binding of proinflammatory cytokines to their cognate receptors, suppress TNF-α mRNA expression, and inhibit monocyte binding and transmigration. Furthermore, in a peritonitis mouse model, KCF18 was able to reduce the infiltration of white blood cells. KCF18 could potentially decrease the risk of vascular inflammation by reducing the release of plasma cytokines and directly acting on the vascular endothelium. This study highlights the potential for combining structure-based in silico design calculations with experimental measurements to develop new anti-inflammatory agents.

Chronic inflammation increases the risk of developing atherosclerosis, restenosis, and arthritis. The initial step in the pathogenesis of atherogenesis is believed to be the injury of the endothelium. Monocytes adhere to activated endothelial cells and migrate across the endothelium, which is a necessary consequence of the inflammatory response in the vasculature. This inflammatory response continues throughout the atherogenic process and is coordinated by interactions between leukocytes and endothelial cells, closely associated with endothelial dysfunction.

The recruitment of leukocytes to the vascular endothelium is dependent on the interplay between E- and P-selectins expressed on the endothelial cell surface and their ligands on leukocytes. VCAM-1 and ICAM-1 play a significant role in this process. Leukocyte activation is a complex process involving the release of several soluble proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, which are important regulators of the inflammatory response in the vessel wall. These cytokines also play a critical role in maintaining host integrity, aiding in white blood cell recruitment to remove invading pathogens and prevent infection.

However, dysfunction in cytokine production can lead to several clinical diseases as mentioned previously. These cytokines can increase endothelial permeability and vasodilation, disrupting the balance between procoagulant and anticoagulant factors. The dysregulation of these cytokines may lead to direct and indirect host injury. Clinical studies have shown that TNF-α and IL-1β levels are significantly elevated in patients with endotoxemia, and that IL-6 levels increase during infectious episodes. TNF-α is a potent inducer of its own gene transcription and has been shown to upregulate the release of IL-1β and IL-6. Additionally, IL-1β is a potent inducer of IL-6 secretion and can increase the expression of several genes.

Chronic bacterial infection can trigger the release of a major cytokine called TNF-α, with a molecular weight of 17.0 kDa, produced mainly by macrophages, lymphocytes, mast cells, monocytes, and fibroblasts. In bacterial or endotoxin-induced shock models, inhibiting TNF-α using anti-TNF-α antibodies can significantly reduce the levels of other cytokines. The TNF-α protein has a β-sandwich structure composed of 10 antiparallel β sheets and can activate two receptors: TNF receptor 1 and 2 (TNFR1 and TNFR2). Another cytokine, IL-1β, with a molecular weight of 17.5 kDa, is mainly produced by macrophages and can induce the upregulation of adhesion molecules on both leukocytes and endothelial cells, leading to a shock-like state. IL-1β is involved in various cellular activities, such as cell differentiation, proliferation, and apoptosis, and its deregulation may cause various autoinflammatory syndromes.

The IL-1β protein can bind to its type I IL-1 receptor (IL-1R) to initiate the IL-1 signal transduction. IL-6, a 20-kDa protein, is also secreted by monocytes, macrophages, endothelial cells, and fibroblasts to stimulate the immune response. The deregulation of IL-6 production has been implicated in several autoimmune diseases, including rheumatoid arthritis, diabetes, depression, and multiple myeloma. The IL-6 protein interacts with the ligand-binding chain IL-6Rα (CD126) and the signal transduction component glycoprotein 130 (gp130), and its nuclear magnetic resonance (NMR) structure was resolved in 1997 (PDB code: 2IL6).

Current therapies aimed at removing inflammatory mediators have not been effective in improving patient outcomes and reducing mortality rates. To address this, new anti-inflammatory strategies are needed. In recent years, peptides have emerged as a promising class of therapeutics for various fields, including neurology, endocrinology, and hematology. Peptides function by binding to specific ligands or cell surface receptors, and advances in bioinformatics and structural databases have enabled the design of novel therapeutic peptides with higher affinity than native proteins for activating or inhibiting signal transduction cascades. Peptide drugs offer numerous advantages, such as predictable metabolism, bioavailability, high efficacy, safety, and tolerability, making them a superior choice over antibodies in many aspects. The use of peptide-based antagonists is a promising approach to develop effective anti-inflammatory or anti-cancer therapies in the future.

In a previous study, a polypeptide derived from IL-8 was truncated to inhibit the binding of IL-8 to CXCR1 based on molecular docking and molecular dynamics simulations. The resulting polypeptide was validated by in vitro SPR detection and cellular assays, and served as a basis for the development of an antagonist peptide that could interfere with the binding of cytokines and cognate receptors as a therapeutic treatment to decrease inflammation severity.

The study focused on designing and synthesizing a potential peptide KCF18, using structural-based molecular docking and MM/PBSA binding free energy calculations. The peptide was then validated in vitro using surface plasmon resonance (SPR) measurements. The study showed that KCF18 effectively decreased the binding of proinflammatory cytokines to their cognate receptors, suppressed TNF-α mRNA expression, inhibited monocyte binding and transmigration, and alleviated the infiltration of white blood cells in a peritonitis model. The findings suggested that KCF18 has strong potential as a therapeutic peptide to reduce the risk of vascular inflammation by reducing plasma cytokine release and acting directly on the vascular endothelium.

Inflammation is a complex biological response to harmful stimuli, and cytokines are key mediators in the inflammatory response. In this study, the researchers aimed to design peptides that could inhibit the binding of proinflammatory cytokines, namely TNF-α, IL-1β, and IL-6, to their cognate receptors, as a potential strategy for the treatment of inflammation. To design the peptides, the researchers investigated the characteristics of each cytokine-receptor complex and selected several amino acids from each receptor on the cytokine-receptor binding interface as potential peptides.

The researchers used molecular docking to analyze the binding of TNF-α to the ectodomain of TNFR1 since the structure of the TNF-α-TNFR1 complex was not available. The binding site of the receptor TNFR1 was determined to contain 18 residues, named SEM18, that had more positive charges at one end. On the other hand, the binding site of TNF-α had more negatively charged residues, which indicated the importance of electrostatic interactions in the binding of TNF-α to the receptor TNFR1.

The structure of the ectodomain of IL-1R complexed with IL-1β was resolved in 1997, and the researchers found that IL-1β is bound to IL-1R at several sites. The binding site of IL-1R was determined to contain more positively charged residues than other sites, and the charge characteristics of IL-1R were similar to those of TNFR1.

The researchers then homology-modeled the N-terminus of IL-6 and used molecular docking to analyze the binding of IL-6 to the ectodomain of the IL-6 receptor. They found that a peptide containing neutral hydrophilic and hydrophobic residues was the best option to compose the binding site. Overall, this study identified potential peptides that could inhibit the binding of proinflammatory cytokines to their cognate receptors, providing a potential strategy for the development of anti-inflammatory drugs.

The initial step of vascular inflammation involves the adherence of monocytes to the endothelial cells, which later infiltrate and differentiate into macrophages. The interaction between monocytes and endothelial cell surface molecules regulates this crucial step. The inflammatory response initiates with the release of TNF-α, which regulates IL-6 and IL-8 levels. IL-1β, another important proinflammatory mediator, acts as an alarm-phase cytokine that triggers various clinical features of inflammation. Inhibition of TNF-α, IL-6, and IL-1β has been suggested to alleviate inflammation exacerbation.

To address this issue, this study aimed to design a composite peptide, KCF18, to bind to all three proinflammatory cytokines simultaneously using molecular docking and MM/PBSA binding free energy calculations. The peptide is composed of 18 amino acids derived from the receptors of the three proinflammatory cytokines. The KCF18 peptide is comprised of six positively charged N-terminal amino acids from TNFR1, six positively charged and hydrophobic middle amino acids from IL-1R, and six neutral C-terminal amino acids from the IL-6 receptor. The theoretical structures of the KCF18-cytokine complexes were compared with the cytokine-receptor structures, indicating that the KCF18 peptide binds to the proinflammatory cytokines in a similar manner to the original binding sites. The electrostatic interactions between the negatively charged region of TNF-α (E104, E107, and E110) and the N-terminal region of KCF18 were found to be the dominant interaction.

The process of vascular inflammation involves the adhesion of monocytes to the endothelium, followed by their infiltration into the endothelial wall and differentiation into macrophages. This crucial step is influenced by the interaction between monocytes and the surface molecules of endothelial cells. During an inflammatory response, TNF-α is initially released, which regulates the levels of IL-6 and IL-8. IL-1β, another important proinflammatory mediator, is an alarm-phase cytokine that can elicit many clinical features of inflammation. Research suggests that inhibiting TNF-α, IL-6, and IL-1β can alleviate inflammation exacerbation.

To this end, a potential composite peptide, KCF18, was designed to bind these proinflammatory cytokines simultaneously using in silico analysis of molecular docking and MM/PBSA binding free energy calculations. The peptide was composed of 18 amino acids derived from the receptors of the three cytokines, with positively charged, hydrophobic, and neutral amino acids strategically placed to enhance binding. The theoretical and in vitro results demonstrated that KCF18 binds to the proinflammatory cytokines in a similar manner to the original binding sites through electrostatic and hydrophobic interactions, with the former being the dominant binding force.

Further, cellular experiments with mutant peptide mKCF18 confirmed that positively charged residues K1, R3, K4, K8, and K10 are critical for the peptide's binding affinity and anti-inflammatory effect. KCF18 was found to suppress cytokine-receptor interactions, cytokine mRNA expression, and cytokine production in endothelial cells and monocytes, resulting in reduced TNF-α secretion. The efficacy of KCF18 was assessed through RT-qPCR and ELISA assay, and the results were compared with those obtained using a TNF-α blocking antibody as an additional condition in the experiments.

In this study, it was shown that KCF18 peptide decreased TNF-α induced TNF-α mRNA expression in both cells in a dose-dependent manner. Conversely, the scramble peptide, mKCF18, did not have an inhibitory effect on TNF-α induced TNF-α mRNA expression. A TNF-α blocking antibody was able to inhibit TNF-α induced TNF-α mRNA expression in a concentration (1 μg/ml) similar to the peptide concentration of 500 nM, while anti-IgG, the negative control of TNF-α blocking antibody, did not have any effect. To assess whether KCF18 could suppress IL-1β induced TNF-α or TNF-α induced IL-1β expressions, an ELISA assay was performed. The results showed that IL-1β induced a 2 fold secretion of TNF-α in THP-1 cells, and KCF18 pretreatment decreased TNF-α expressions. However, mKCF18 could not inhibit IL-1β induced TNF-α expression. TNF-α blocking antibody dramatically suppressed IL-1β induced TNF-α expression. On the other hand, KCF18 also decreased TNF-α induced IL-1β expressions in a dose-dependent manner, and the TNF-α blocking antibody suppressed TNF-α induced IL-1β expression. KCF18 was also found to reduce the adherence of THP-1 to cytokine-activated endothelial cells and suppress endothelial transmigration of THP-1, as well as inhibit TNF-α-mediated p65 nuclear translocation and endothelial ICAM-1 expression. In an animal model of peritonitis, KCF18 was found to inhibit white blood cell infiltration, demonstrating its anti-inflammatory properties.

Various therapies have been developed to reduce inflammation after bacterial infections, but these agents have not been successful in clinical trials. To address this issue, novel therapeutic approaches are needed that can decrease inflammation and improve the immune response to cytokines. Peptide inhibitors have the potential to be more specific than small-molecule inhibitors, but their delivery to cells and stability in live organisms remain a challenge. Our peptide, KCF18, was designed to interfere with cytokine-receptor interactions on cell surfaces and prevent leukocyte infiltration in a peritonitis model. Our data revealed a new role for KCF18 in targeting the receptors of three cytokines and regulating inflammatory responses in vitro and in vivo. Our results suggested that peptide-mediated inhibition of multiple cytokine-induced immune responses could offer a unique opportunity to develop urgently needed therapeutic interventions for inflammation-related diseases. KCF18 may serve as a novel immunomodulatory therapeutic agent and can be utilized to design and discover new therapeutic drugs for treating inflammation. Computational simulations have also shown that this peptide is a useful tool for developing anti-inflammatory peptides. However, there may be a discrepancy between physiological cell responses and molecular biological assays due to the number of receptors not always correlating with the magnitude of the response.

Posted in: Anti-Inflammatory

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