Fig. 4: KSI-6666 is a pseudoirreversible inhibitor.

a Schematic figure of functional reversibility assay. b Functional reversibility of KSI-6666 and W146 examined by agonist-induced S1PR1 internalization assay. HEK293 cells expressing HiBiT-tagged human S1PR1 were treated with each compound and concentration-dependent S1PR1 internalization induced by FTY720-P or S1P, represented by luciferase activity, was measured. Calculated E_max values for the FTY720-P and S1P-induced response on treatment with each compound at different concentrations are summarized in the right graphs. Data for the upper and lower graphs were obtained from five and three independent experiments, respectively. Statistical analysis was performed by one-way ANOVA, followed by Tukey’s test for KSI-6666. Results are expressed as the mean ± s.e.m. c Functional reversibility of KSI-6666 and W146 examined by agonist-induced GTP binding assay. Calculated E_max values for the FTY720-P-induced response on treatment with each compound at different concentrations. Data were obtained from eight independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Tukey’s test for KSI-6666 (control, n = 8; 1 nM, n = 4; 10 nM, n = 4; 100 nM, n = 5; 1 µM, n = 4 biological replicates; mean ± s.e.m.). d Dissociation kinetics of KSI-6666 from S1PR1. KSI-6666 (30 nM) or W146 (10 µM) was incubated with cells expressing human S1PR1 for 60 min. After the removal of compounds through a washing process, cells were allowed to incubate for an indicated period to facilitate the dissociation of compounds from S1PR1. The cells were stimulated with 10 nM Merck S1PR1 agonist, and Ca²⁺ mobilization was induced. The typical Ca²⁺ mobilization responses are shown in the left panels. The relative response was plotted against the incubation time in the right figure, and the complex half-life time was evaluated. The results obtained from the operation without washout were plotted at 0 h. Data on KSI-6666 and W146 was obtained from four and three independent experiments, respectively. e Pharmacokinetics of KSI-6666 in rats. KSI-6666 was intravenously administered to rats, and blood samples were chronologically collected. Results are expressed as the mean ± s.e.m (n = 3 rats). Source data are provided as a Source Data file.