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Screening and Mechanism of Antagonist Peptide for CC Chemokine Receptor 1 (CCR1) Derived from Viral Macrophage Inflammatory Protein II Liu, Sha; Ding, Qing; Wei, Pijin; Sun, Hanxiao; Li, Xiuying; An, Guijie; Yang, Yan & Zhou, Jingguang Abstract Purpose: To search for effective antagonist peptide of CC chemokine receptor 1 (CCR1), and evaluate the potential role and mechanism of peptide C18P derived from viral macrophage inflammatory protein II (vMIP-II). Methods: Alignment, simulated peptide-cut, bioinformatics and protease digestion were used to screen and prepare antagonist peptide. Interactions between C18P and CCR1 were determined by radioligand binding assays and [35S]GTPγS binding experiment. Chemotaxis assay was utilized to assess the potency for inducing or inhibiting peripheral blood mononuclear cells (PBMCs) migration. Ligand- induced intracellular calcium mobilization was tested by flow cytometry. Results: A peptide containing 18 amino acids (C18P) was screened. C18P bound to CCR1 with a Kd of 5.7 ng/ml and displaced 125I-labeled MIP-1α and 125I-labeled RANTES on human CCR1-transfected HEK293 cells (IC50 = 11.2 and 9.6 ng/ml, respectively) in radioligand binding studies. C18P lacked intrinsic agonist activity but strongly inhibited HCC-1 (100 nM) induced [35S]GTPγS binding (IC50 = 3.7 ug/ml), chemotaxis induced by HCC-1, MIP-1α and RANTES (IC50 = 23, 25 and 13.1 ng/ml, respectively), and intracellular calcium mobilization. Conclusion: These results demonstrate that bioinformatics and protease digestion are feasible to screen and prepare C18P, and that C18P is a novel and specific small molecule peptide antagonist of CCR1 with therapeutic potential for preventing cell migration. Keywords CC Chemokine receptor 1; Simulated peptide-cut; Antagonist peptide; Viral macrophage inflammatory protein II; Bioinformatics; Protease digestion; HEK293 cells; Radioligand binding

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