Abstract This is a revised competitive renewal application for a NIH R01 grant entitled “Structures and Interactions of Chemokine Receptors” that has been continuously funded and very successful for 22 years. The long- standing goals of this project are to understand the mechanisms of chemokines and their receptors in various pathologies and to translate this information into the development of new intervention strategies. During the past funding period, we have made significant progress toward these goals. Specifically, we have de novo designed and chemically synthesized novel agonist molecules of CXCR4 receptor and completed a series of studies to characterize their in vitro and in vivo biological activities in eliciting site-specific migration and distribution of human neural stem cells and exerting significant and selective therapeutic effect in mice with neurodegenerative disease. The molecular mechanisms of action of these agents have been analyzed with a panel of point mutations at 24 residues located in each of the seven transmembrane domains of CXCR4 to map critical sites for CXCR4-agonist recognition and signaling. To further advance these peptide agonists toward potential therapeutics, we have developed a new strategy for peptide optimization with a small molecule mimicry approach. A novel small molecule with potent biological activity in itself has been discovered to mimic and to replace a large portion of the peptide agonist, resulting in a new prototype small molecule- peptide conjugate agonist with much higher CXCR4 affinity, smaller size, and lower synthetic cost that are advantageous for therapeutic development in the new grant period. These exciting discoveries and results have laid a solid foundation for continuing translational research in regenerative medicine and basic research to understand the mechanisms underlining stem cell migration and signaling.