Molecular Dynamics and Free Energy Calculations Predict Binding Mode and Affinity Determinants of Specialized...

GPR101 is an orphan G protein-coupled receptor (GPCR) with unusually high constitutive activity and has recently emerged as a target for specialized pro-resolving mediators (SPMs), endogenous lipids that actively terminate inflammation and promote tissue repair. Given the therapeutic relevance of pro-resolution signaling in chronic pain and inflammatory disorders, understanding how SPMs engage GPR101 is of great significance. Although cryo-EM structures suggest an occluded orthosteric cavity, SPMs such as RvD5_n‑3 DPA are potent agonists, creating uncertainty about their binding modes. Long-timescale molecular dynamics (MD) simulations, MM-GBSA per-residue energy decomposition, residue-interaction network analysis, and alchemical relative binding free-energy (RBFE) calculations were used to predict interactions between SPMs with GPR101. MD trajectories revealed a stable RvD5_n‑3 DPA pose beneath an extracellular loop, stabilized by M184, W186, and Y415. Transmembrane distance metrics across five independent 1 μs MD simulation trajectories showed persistent stabilization of an active-like state even without modeled G-protein coupling. RBFE analyses quantified the scaffold-dependent effects of C17 alcohol stereochemistry, oxidation, methylation, and 3-oxa substitution. A double mutant cycle calculation identified a coupling between C17 alcohol and residue M184. Novel dual-modified analogs were computationally predicted to retain high affinity while improving metabolic stability. Benchmarking demonstrated that membrane-free thermodynamic integration (AMBER) yielded accurate, low-variance results with shorter wall time than membrane-inclusive replica exchange (NAMD). These results provide the first atomistic model of SPM binding to GPR101 and establish an RBFE-guided framework for designing next-generation pro-resolving mediator analogs with enhanced pro-resolving effects and stability.