Antagonist Peptide Design Of CCR1Based On VMIP-Ⅱ Simulated Peptide-cut And It’s Bioactivity Study

PurposeTo design and screen antagonist peptide of CCR1derived from vMIP-II, which is abroad-spectrum chemokine antagonist. Bioinformatics methods are based on the high structuralsimilarity of chemokine family. Bioactivity of peptide C18P was evaluated.MethodAfter sequence alignment, proteases were utilized to the simulated peptide-cut of vMIP-II.Peptide fragments including the conserved residues were picked out and analyzed. Recombinantplasmid pcDNA4/TO-CCR1was transfected into HEK293by using liposome transfectionmethod. HEK293/CCR1cells expressing CCR1acted as the research platform of peptide C18P.[35S] GTPγS binding experiment was performed to detect the function of CCR1and the ability toactivate receptor of C18P. Chemotaxis assay and calcium mobilization experiment were appliedto detect the ability of C18P to inhibit cell migration and elevation of Ca2+concentration inducedby CCR1ligands. Ligand-receptor binding experiment was utilized to determin binding affinityof C18P. Model of inflammatory cell induced by endotoxin was used to assess the effect of C18Pon PBMC from the perspective of molecular level.Result(1) HEK293/CCR1cells can successfully efficiently express CCR1and have normalreceptor function. CCR1was stimulated by MIP-1α with EC50of5.57ng/ml.(2) Peptide C18P derived from vMIP-II is a specific antagonist of CCR1and have nochemotaxis. Chemotaxis induced by HCC-1, MIP-1α and RANTES were strongly inhibited byC18P in a concentration-dependent manner, with IC50values of19.08ng/ml,21.27ng/ml and10.56ng/ml, respectively. It can block the calcium mobilization induced by HCC-1, MIP-1α andRANTES.(3) Ligand-receptor binding experiment showed that the peptide C18P completely inhibited125I-MIP-1α binding to CCR1with IC50value of14.63ng/ml. Inhibition rate was80%when ligand concentration was100ng/ml with IC50value of15.09ng/ml.[35S] GTPγS bindingexperiments further demonstrated the antagonist role, not agonist, of C18P.(4) ELISA detection results demonstrated that compared with the LPS group, middle dosegroup (5ng/ml) and high dose group (15ng/ml) of C18P and dexamethasone could effectivelyreduce TNF-α, IL-6and IL-8expression. The results of Q-PCR indicated that compared withLPS group, C18P group and dexamethasone group could effectively reduce the mRNAexpression of TNF-α, IL-6and IL-8. NO detection results showed that compared with LPS group,the concentration of NO was significantly decreased in peptide C18P group and dexamethasonegroup.ConclusionThis study demonstrates that peptide C18P derived from vMIP-II is an antagonist of CCR1.It inhibits chemotaxis, calcium mobilization, and G protein activation induced by ligands, andhas good affinity with CCR1. It also inhibits the inflammatory cells secreting inflammatorycytokines, supporting that it can be developed to be an anti-inflammatory drug in the future.