Investigation the Possibility of Using Peptides with a Helical Repeating Pattern of Hydro-Phobic and Hydrophilic Residues to Inhibit IL-10

Abstract

Blockade of IL-10 signalling clears chronic viral and bacterial infections. Immunization together with blockade of IL-10 signalling or relatively low level of IL-10 further enhances viral and bacterial clearance. IL-10 functions through binding to interleukin 10 receptor (IL-10R). Here we showed that peptides P1 and P2 with the hydrophobic and hydrophilic pattern of the IL10R-binding helix in IL-10 could bind with either IL-10R1 or IL-10, and inhibit inflammatory signals with long duration and negligible cytotoxicity in vitro. Furthermore, P2 can enhance antigen specific CD8+ T cell responses in mice induced by the vaccine based on a long peptide of protein E7 in a human papillomavirus type 16.

Inhibitory mechanism of peptides with a repeating hydrophobic and hydrophilic residue pattern on interleukin-10

“…In a previous study, 1 it was shown that P1 and P2 compete with IL-10 in binding with IL-10R1 (Fig. S2).…”

“…1 No multiplycharged adduct peaks, formed between P1/P2 and IL-10R1, were detected using MALDI mass spectrometry, and no corresponding peak was identified in the case of P3 (negative control). Thus, to further study the potential for the interplay of bioactive peptides P1 and P2 with IL-10R1, molecular dynamics (MD) simulation was used to model the complexes formed, and compared to the negative control peptide P3.…”

“…This was in accordance with our previous report that P1 or P2 can prevent IL10 from binding to immobilized IL10-R1, and no competitive binding was observed for P3. 1 MD simulation confirms P1 or P2 binds to IL-10R1 with higher affinity than IL-10 The representative structures of 1:1 complexes P1/IL-10R1 and P2/IL-10R1 (shown in Fig. S3C and S4C) were selected to evaluate the interaction mechanism.…”

“…These results meant that IL-10R1 would bind preferentially to P1 or P2 rather than IL-10, further supporting the SPR assay result that P1 and P2 compete with IL10 in binding to immobilized IL10-R1. 1 In vitro inhibition of IL-10 activity by truncated and mutated P1 and P2 peptides: the key structural characteristics of inhibiting peptides IL-10 is known to negatively regulate antigen presentation cells secreting IL-12; if IL-10 signaling is blocked, antigen presentation cells will produce more IL-12, which is critical to mount a T helper 1 response. Strong Th1 responses correlate with viral clearance and tumor rejection.…”

“…24 We proposed a way for designing IL-10 inhibiting peptides based on a helix segment of IL-10 within IL-10/IL-10R1 interaction region, and demonstrated that peptides with repeating pattern of hydrophobic and hydrophilic residues are able to inhibit IL-10 both in vitro and in vivo. 1 We have confirmed that the peptides are able to bind IL-10R, so as to inhibit the signaling of IL-10 with high efficiency supported by the following assays 1 :-i) Surface Plasmon Resonance (SPR) spectral analysis suggested the peptides displayed a level of binding affinity at sub micro-molar level with IL-10R, and in addition their binding competed with IL-10; ii) an MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay of mouse mast cell MC/9 cells indicated that the peptides counteracted the effect of IL-10 stimulation and limited cell proliferation, iii) the peptides inhibited IL-10 secretion, confirmed by ELISA using LPS-stimulated human PBMCs and macrophage U937 cell lines; iv) the peptides competed with anti-IL-10R antibodies for binding with IL-10R1 while showing negligible cytotoxicity; and v) one peptide enhanced vaccine induced antigen specific CD8+ T cell responses. These results indicated that the peptides are functional, with in vivo bioactivity, and potentially may block IL-10 signaling in human.…”