Determining binding sites of cytokine–cognate receptor complexes

Three proinflammatory cytokines, namely TNF-α, IL-1β, and IL-6, which play a critical role in the inflammatory response, were selected to analyze the binding sites of cytokine–cognate receptor complexes. To design the preferable peptides for binding these three cytokines, the characteristics of each cytokine–receptor complex was first investigated. Subsequently, several amino acids from each receptor on the cytokine–receptor binding interface were selected as the potential peptides to compose a new peptide for inhibiting inflammation. In the absence of the structure of the TNF-α–TNFR1 complex, molecular docking was performed for the binding of TNF-α to the ectodomain of TNFR1. The binding site of the receptor TNFR1 was determined to contain 18 residues, named SEM18 (63SENHLRHCLSCSKCRKEM80) that imparted more positive charges at one end (Supporting Figure S1A). The binding site of TNF-α was more negatively charged, indicating that electrostatic interactions are crucial for the binding of TNF-α to the receptor TNFR1. The structure of the ectodomain of IL-1R complexed with IL-1β was resolved in 199749 (PDB code: 1ITB), which showed 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 (108QAIFKQKLPVAGD120) than other sites, and the charge characteristics of IL-1R were similar to those of TNFR1 (Supporting Figure S1B). In the absence of the structure of the N-terminus of IL-6, the structure was homology modeled from the Phyre2 web server to form a new IL-6 structure with N-terminus, which was equilibrated for 50-ns MD simulations. According to the docking of IL-6 to the ectodomain of the IL-6 receptor, a peptide containing neutral hydrophilic and hydrophobic residues (162VDYSTVYFVN171) was determined to compose the binding site (Supporting Figure S1C).

Interactions between cytokines and composite peptide

Based on the determined binding sites of the respective cytokine–cognate receptor complex, a new composite peptide (1KCRKEMFKQKLPYSTVYF18, called KCF18), which contained five positively charged residues and six hydrophobic residues, was designed to bind to the three cytokines simultaneously. Analysis of the surface charge and lipophilicity distributions of the composite peptide revealed that more positively charged residues were present at one end, and more hydrophobic residues were distributed at the other end. Thus, the peptide exhibited amphiphilic properties (Fig. 1). The composite peptide KCF18 was redocked to the three cytokines, showing that the predicted binding site of KCF18 for TNF-α was near the original binding site of the TNF-α–TNFR1 complex. In the predicted binding mode, the positively charged residues of KCF18 (K1, R3, K4, K8, and K10) formed strong electrostatic interactions with the negatively charged residues of TNF-α (E104, E107, and E110) (Fig. 2A). The neutral hydrophobic residues of KCF18 also bound to the hydrophobic residues of TNF-α (Fig. 2B). In the KCF18–IL-1β complex, the redocked binding site was slightly different from the original binding site of the IL-1β–IL-1R complex. In the redocked binding site, the positively charged residues of KCF18 (K1, R3, K4, K8, and K10) were bound to the negatively charged residues of IL-1R (Fig. 2C). The hydrophobic residues of KCF18 (L11 and P12) formed hydrophobic interactions with the hydrophobic residues of IL-1β (F150 and V151) (Fig. 2D). In the KCF18–IL-6 complex, the redocked binding site was near the original binding site of the IL-6–IL-6 receptor complex. The positively charged residues of KCF18 (K1, R3, and K4) were bound to the negatively charged residues of IL-6, and its hydrophobic residues (L11, P12, V16, and F18) also interacted with the hydrophobic residues of IL-6 (A13, A14, M118, V122, and L123) (Fig. 2E,F).

Figure 1 Surface property analysis of the designed peptide KCF18. (A) Surface charge distribution of KCF18 was calculated using the Poisson–Boltzmann equation. Blue color corresponds to positive and red color to negative electrostatic potential. KCF18 is more positively charged at one end. (B) Surface lipophilicity distribution of KCF18, in which blue color represents hydrophilic residues, whereas green color represents hydrophobic residues. It seems that more hydrophobic residues are distributed at the far end. Full size image

Figure 2 Surface property analysis of preferable binding poses for KCF18 to proinflammatory cytokines. KCF18 is represented as an orange-colored and green-colored loop structure. Residues around the binding interface are labeled and are shown as sticks. Surface charge distribution of KCF18 binding to cytokines was calculated using the Poisson–Boltzmann equation. Blue color corresponds to positive and red color to negative electrostatic potential. (A) KCF18–TNF-α, (C) KCF18–IL-1β, and (E) KCF18–IL-6 complexes. Surface lipophilicity distribution of KCF18 binding to cytokines, in which blue color represents hydrophilic residues, whereas green color represents hydrophobic residues. (B) KCF18–TNF-α, (D) KCF18–IL-1β, and (F) KCF18–IL-6 complexes. Full size image

Binding free energy calculations for the binding of composite peptide to cytokines

To obtain more insights on the molecular docking of the composite peptide to the three cytokines and to determine the dominant interactions between the KCF18 peptide and the cytokines, MM/PBSA binding free energy calculations for the binding of KCF18 to the cytokines were performed, as shown in Supporting Figure S2A. To reduce errors due to limited sampling in the MD simulations, the various complex systems were repeated two times with different initial velocities to perform the first 200 ns MD simulations, indicating that the root-mean-square deviation (RMSD) values of the complex backbone atoms of repeated systems were similar and fluctuated to reach a plateau after 100 ns MD simulations (Supporting Figure S2B). The KCF18 peptide RMSD profiles indicated that the peptide may adopt a stable conformation in all systems after 130 ns MD simulations (Supporting Figure S2C). The binding free energy (ΔG bind ) for the binding of the composite peptide KCF18 to the cytokine TNF-α (−583.6 ± 3.6 kJ/mol). The binding free energies for the KCF18–IL-1β and KCF18–IL-6 complexes were −394.9 ± 3.8 and −526.6 ± 5.3 kJ/mol, respectively. The computed binding differences are much larger than the corresponding experimental relative affinities, but indicate that the two cytokines had a higher binding affinity with KCF18. When KCF18 was bound to these cytokines, electrostatic interactions gave the dominant contribution to the computed MM/PBSA binding affinities whereas the contribution due to VDW interactions was minor (Supporting Figure S2A). Moreover, a truncated peptide SEM18 truncated from the binding site of the receptor TNFR1 was also redocked to cytokine TNF-α to calculate MM/PBSA binding free energy (−144.60 ± 4.8 kJ/mol), which is a much higher energy than that of KCF18 to TNF-α complex. This finding implied that the designed peptide KCF18 theoretically has much better binding affinity than the truncated peptide SEM18.

SPR measurements for the association between composite peptide and cytokines

To confirm whether KCF18 binds to the three cytokines, the composite peptide KCF18 was synthesized for SPR measurement by using a Biacore T200 instrument. SPR sensorgrams revealed a positive change in RUs. This finding revealed that KCF18 could bind to the cytokines (TNF-α and IL-6) immobilized on the CM5 sensor chip (Fig. 3). The measured response for the binding of KCF18 to TNF-α also increased when the concentration of KCF18 increased, indicating that binding occurred in a concentration-dependent manner (Fig. 3A). When KCF18 was injected, KCF18 rapidly bound to TNF-α, and the curve plateaued after a few seconds. Thereafter, KCF18 dissociated rapidly during the rinsing of the chip with buffer. For a steady–state interaction, the binding isotherm was generated to determine the equilibrium dissociation constant K D (60.9 μM) and R max (197.1 RU) for the binding of KCF18 to TNF-α (Fig. 3B). The measured response for the binding of KCF18 to IL-6 also increased with the KCF18 concentration, similar to the profile for the binding of KCF18 to TNF-α, as shown in Fig. 3C. The kinetic analysis of the binding isotherm was also performed to determine the equilibrium dissociation K D (111.5 μM) and R max (210.3 RU) for the binding of KCF18 to the cytokine IL-6 (Fig. 3D). In addition, the measured response for the truncated peptide SEM18 to TNF-α increased with the SEM18 concentration, similar to the profile for the binding of KCF18 to TNF-α, as shown in Supporting Figure S3. The kinetic analysis of the binding isotherm was also performed to determine the equilibrium dissociation K D (68.3 μM) and R max (8.2 RU) for the binding of SEM18 to the cytokine TNF-α. Because it was extremely difficult to immobilize IL-1β on the sensor chip, the interaction between KCF18 and IL-1β was not measured through SPR detection but was confirmed through cellular assays and the animal model.

Figure 3 SPR analysis for KCF18 binding to proinflammatory cytokines. (A) KCF18 was injected over TNF-α immobilized on the CM5 sensor chip. As the concentration of KCF18 increased, the measured response for the binding of KCF18 to TNF-α also increased, indicating that binding was concentration dependent. (B) For the steady–state interaction, a binding isotherm was generated to determine the equilibrium K D and R max for the binding of KCF18 to cytokine TNF-α, which were found to be 60.9 μM and 197.1 RU, respectively. (C) KCF18 was injected over IL-6 immobilized on the CM5 sensor chip. The measured response for the binding of KCF18 to IL-6 also increased with the concentration of KCF18, similar to the profile of the binding of KCF18 to TNF-α. (D) The kinetic analysis of binding isotherm was also performed to determine the equilibrium dissociation K D and R max for KCF18 binding to cytokine IL-6, which were found to be 111.5 μM and 210.3 RU, respectively. Full size image

Composite peptide binding to the cytokines for inhibition of cytokines induced monocyte binding and transmigration to endothelial cells

To confirm that the in silico designed composite peptide binds to the proinflammatory cytokines, peptides at different concentrations (500 nM, 50 nM, and 5 nM) were incubated with the three cytokines to examine whether the peptide inhibits the adherence of THP-1 cells (human acute monocytic leukemia cell line) to cytokine-activated human microvascular endothelial cells (HMEC-1). The activation of HMEC-1 by TNF-α, IL-1β and IL-6 leaded to important enhancement in the count of stuck THP-1 on the HMEC-1 monolayer. Meanwhile, the pretreatment of HMEC-1 with KCF18 repressed the count of THP-1 cells sticking to various cytokine-treated HMEC-1 cells, with an almost 100% (P < 0.001) decrease (Fig. 4). Thus, KCF18 could inhibit cytokine-induced monocyte binding. This finding indicated that the peptide suppressed the association between the cytokines with the cell surface receptors. To verify whether the suppression is caused by the peptide KCF18, a random peptide with 25 amino acids (CF25; 1CPLNGSTVYGHLRHCLSCSGTMVKF25) and a truncated peptide SEM18 derived from TNFR1 were synthesized for monocyte binding assays. The results showed that the nonrelated peptide CF25 could not repress cytokine-increased monocyte binding to HMEC-1 cells and peptide SEM18 could only reduce TNF-α-induced monocyte binding, but not cytokines IL-1β, and IL-6 (Fig. 4D and Supporting Figure S4). Furthermore, transmigration assays were performed to examine whether KCF18 inhibits cytokine-induced monocyte transmigration. The numbers of transmigrating THP-1 cells were significantly increased when endothelial cells were cultured with the cytokines (TNF-α, IL-1β, and IL-6) compared with the migration in the absence of cytokines. Treatment with the designed peptide KCF18 could reduce the number of transmigrating THP-1 cells induced by the cytokines (Fig. 5). The result of the transwell assay was similar to that of the monocyte binding assay. Thus, KCF18 could inhibit the binding of the cytokines to their receptors on the cell surface. Therefore, the three cytokines could not transmit the inflammatory signal to cells for regulating immune responses.

Figure 4 KCF18 inhibits various cytokine-induced monocyte adhesion to HMEC-1. HMEC-1 cells were pretreated with various concentrations of KCF18 or (D) peptide CF25 for 1 hour and were then stimulated with 20 ng/mL (A) TNF-α, (B) IL-1β, or (C) IL-6 for 18 hours. Adhesion of fluorescent THP-1 cells was photographed by fluorescent microscopy, and fluorescence intensity was calculated. “Control” cells were only incubated with the culture medium (without peptides). Values are mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 as compared with control; #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 as compared with cells stimulated with cytokines in the absence of peptides. Full size image

Figure 5 KCF18 inhibits cytokine-induced transmigration of monocytes. HMEC-1 cells were pretreated with various concentrations of KCF18 for 1 hour and then stimulated with 20 ng/mL (A) TNF-α, (B) IL-1β, or (C) IL-6 for 18 hours; thereafter, THP-1 cells were allowed to transmigrate through the HMEC-1 monolayer. Values are mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 as compared with control; #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 as compared with cells stimulated with cytokines in the absence of KCF18. Full size image

To evaluate whether the inhibitory effects of the composite peptide KCF18 on THP-1 binding and endothelial transmigration were as a result of the cytotoxic effects on endothelial cells, HMEC-1 cells were treated with various concentrations of KCF18. The viability of HMEC-1 cells was assessed by the WST-1 assay after 24 and 48 hours treatment. As described in Supporting Figure S5, no important discrepancy was noticed in cell viability among control cells and peptide-treated cells. This result suggested that endothelial cytotoxicity is not the reason for the repression of THP-1 adherence and endothelial transmigration by KCF18.

Inhibitory effects of composite peptide KCF18 on inflammatory cytokine-induced TNF-α transcription

We used qPCR assays to quantify the effects of KCF18 on TNF-α-, IL-6-, and IL-1β-induced TNF-α mRNA expression. Compared with control cells, TNF-α mRNA expression levels were increased in HMEC-1 (Fig. 6A–C) and THP-1 cells (Fig. 6D–F) cultured with TNF-α. The negative control peptide CF25 had no inhibitory effect on TNF-α-induced TNF-α mRNA expression (Fig. 6A). However, peptide KCF18 significantly reduced TNF-α-mediated TNF-α mRNA expression. In addition, no significant difference was observed in mRNA expression levels between peptide-treated cells and control cells (Fig. 6A). Similarly, KCF18 suppressed IL-6- and IL-1β-induced TNF-α mRNA expression. This finding indicated that KCF18 interfered with the binding of these cytokines to their receptors and further reduced downstream TNF-α mRNA transcription (Fig. 6B–F).

Figure 6 Peptides KCF18 and CF25 affect the expression of cytokine-induced TNF-α in HMEC-1 and THP-1 cells. TNF-α mRNA levels induced by TNF-α (A and D), IL-1β (B and E), or IL-6 (C and F) in HMEC-1 (A–C) and THP-1 (D–F) were determined using qPCR assays, as described in Materials and Methods. GAPDH cDNA was used as an internal control. Values are mean ± SD of mRNA levels relative to those for GAPDH from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 as compared with control; #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 as compared with cells stimulated with cytokines in the absence of the peptides. Full size image

Anti-inflammatory effects of KCF18 in a peritonitis model in vivo

Neutrophils are rapidly mobilized from the bone marrow into the blood during acute inflammatory reactions. Intraperitoneal injection of thioglycollate elicits a robust influx of neutrophils into peritoneal cavity50,51. We assessed the effects of KCF18 on inflammatory cell recruitment by using a thioglycollate-induced acute peritonitis model. At 24 hours after thioglycollate stimulation, elicited inflammatory cells were detected in the peritoneal cavity. Administration of KCF18 at 4 hours after thioglycollate stimulation significantly reduced total white blood cell infiltration in the peritoneal cavity. However, the negative control peptide CF25 had no potential anti-inflammatory effect (Fig. 7). As estimated, the influence of mKCF18 on reducing of WBC infiltration is worse than KCF18 peptide (Supporting Figure S9).