Design, molecular characterization and therapeutic investigation of a novel CCR8 peptide antagonist that attenuates acute liver injury by inhibiting infiltration and activation of macrophages

During liver injury, intrahepatic macrophage compartment is augmented by circulating monocytes that infiltrate the liver driven by C-C motif chemokine ligand/C-C motif chemokine receptor (CCL/CCR) axis including CCL1‒CCR8 axis, thereby contributing to liver inflammation. Numerous small molecular receptor antagonists, including R243, have been developed for targeting CCR8; however, these agents face challenges in clinical translation, potentially attributed to their poor pharmacokinetic profiles, lack of target specificity, and potential adverse effects. In this study, we designed four CCR8 antagonizing peptides (AP8i-AP8iv) and performed molecular characterization <i>in silico</i> and therapeutic investigation <i>in vitro</i> and <i>in vivo</i>. Based on <i>in silico</i> docking, molecular dynamic simulation using homology build model and <i>in-vitro</i> (competitive) binding studies, AP8ii (YEWRFYHG) evidenced highly favorable and selective interactions at the CCR8-active site. AP8ii inhibited CCL1-driven chemotaxis and LPS/IFN<i>γ</i>-induced pro-inflammatory activation of monocytes-macrophages <i>in vitro</i>. In a CCl₄-induced acute liver injury mouse model, AP8ii treatment decreased intrahepatic infiltration of circulating monocytes. Moreover, AP8ii reduced liver inflammation, as indicated by decreased F4/80, IL6 and iNOS expression, diminished ALT levels, and attenuated fibrosis, as indicated by reduced collagen-I expression. In conclusion, we report a novel CCR8-antagonizing peptide that inhibited CCL1-driven intrahepatic monocytes infiltration and differentiation into pro-inflammatory phenotype, consequently ameliorating liver inflammation and fibrogenesis in an acute liver injury mouse model.