Computational Insights into the Activation Mechanism of CXCR4: Implications for the Design of Small Molecule Agonists

G protein-coupled receptors (GPCRs) are transmembrane proteins that mediate diverse signaling functions, making them important therapeutic targets. The chemokine receptor CXCR4, a GPCR, plays multifaceted roles in both normal physiological and pathological processes. Here, we constructed conformational free energy profiles of CXCR4 activation using Targeted Molecular Dynamics (TMD) and Molecular Dynamics (MD) simulations combined with our refined Coarse-Grained (CG) model for membrane proteins. The simulations revealed that CXCR4 activation involves three distinct transition states. TS1, which exhibits the highest activation energy barrier, primarily involves conformational changes in intracellular loop 3 (ICL3) and the prerearrangement of transmembrane helices TM5 and TM6. Additionally, the stabilization of the specific active conformations of W94 and E288 within the CXCR4 active site was found to reduce the activation energy barriers of TS2 and TS3, respectively. Alanine scanning further revealed the dynamic roles of other residues whose putative crucial role in CXCR4 activation had previously been postulated, transitioning from stabilizing the inactive state to facilitating activation in later stages. Guided by these computational insights, we designed a small-molecule compound, HL82624, using a dual-moiety strategy combining the first two amino acids of SDF-1α (Lys and Pro) with the CXCR4 antagonist HF51116. Competitive binding and cell migration assays showed that HL82624 binds to CXCR4 and effectively triggers cell migration, confirming its activity as a CXCR4 agonist. Taken together, this study provides mechanistic insight into CXCR4 activation and a computational strategy for the rational design of small-molecule CXCR4 agonists.