Label-free cell phenotypic profiling of sphingosine-1-phosphate receptor 1 and discovery of its agonist from natural products.

Sphingosine-1-phosphate receptor 1 (S1PR1) belonging to G protein-coupled receptors (GPCRs), plays a significant role in autoimmune diseases and cancer via regulating immune cell migration, vascular barrier function, and cell survival. Although four S1PR1 agonists have been clinically approved, their applications are limited by adverse effects, necessitating the discovery of novel S1PR1 agonists. In this study, we established a HEK293T-hS1PR1 high-throughput screening model for S1PR1 ligand discovery using dynamic mass redistribution (DMR) and fluorescent imaging plate reader (FLIPR) assays. Through structure-based virtual screening of anti-inflammatory compounds, we identified corylin, a natural compound derived from Psoralea corylifolia L., as a novel S1PR1 agonist. Corylin demonstrated micromolar-level agonistic activity with EC50 values of 7.50 μM (95% CI: 6.46-8.75 μM) in DMR assay and 5.99 μM (95% CI: 2.16-16.64 μM) in FLIPR assay. Notably, corylin showed no activity against two other GPCRs (GPR84 and GPR120) commonly co-expressed with S1PR1 in immune cells. Molecular docking indicated that corylin interacted with S1PR1 via hydrophobic interaction, lacking of π-stacking with Trp269, a key interaction for SEW2871. Subsequent 500-ns molecular dynamics simulations revealed a ligand reorientation and the formation of a π-π interaction with Trp269, observed with ~ 50% occupancy, thereby demonstrating the stable binding of corylin to S1PR1. Furthermore, pathway deconvolution analysis confirmed that corylin activated PI3K/Akt and MEK/ERK downstream signaling cascades. This study identifies corylin as a novel S1PR1 agonist with potential for drug design and presents a robust high-throughput screening method for S1PR1 agonist discovery.