Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases.A natural fragment of serum albumin,named EPI-X4,has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases.To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4.Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4.The EPI-X4 derivative JM#21 bound CXCR4 and sup-pressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100.EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and sup-pressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model.Moreover,topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis.Thus,rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis.Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis,asthma and other CXCR4-associated diseases is highly warranted.