Pseudoirreversible inhibition elicits persistent efficacy of a sphingosine 1-phosphate receptor 1 antagonist

Sphingosine 1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor, is required for lymphocyte trafficking, and is a promising therapeutic target in inflammatory diseases. Here, we synthesize a competitive S1PR1 antagonist, KSI-6666, that effectively suppresses pathogenic inflammation. Metadynamics simulations suggest that the interaction of KSI-6666 with a methionine residue Met124 in the ligand-binding pocket of S1PR1 may inhibit the dissociation of KSI-6666 from S1PR1. Consistently, in vitro functional and mutational analyses reveal that KSI-6666 causes pseudoirreversible inhibition of S1PR1, dependent on the Met124 of the protein and substituents on the distal benzene ring of KSI-6666. Moreover, in vivo study suggests that this pseudoirreversible inhibition is responsible for the persistent activity of KSI-6666.

Supplementary Figure 2. In vivo effect of KSI-6666 administration on normal mouse and mouse disease model.a. Histological findings in lungs of rats after repetitive oral administration of test compounds for 4 weeks.The test compound dissolved or suspended in 0.5% methyl cellulose 400 solution was orally administered to 8-week-old male Wistar Hannover rats for 4 weeks.KSI-6666 was administered at 0.05, 0.5, and 5 mg/kg b.i.d., and FTY720 at 5 mg/kg q.d.Following euthanasia, lungs were collected and histologically examined (n = 5 rats /group).N: no finding; Y: finding present; −: not remarkable/not present; ±: minimal; 1+: slight; 2+: Moderate; 3+: Marked.b.Lung weight of rats after repetitive oral administration of test compounds for 4 weeks.
The collected lungs were weighed in the rat study shown in Supplementary Figure 2a.Statistical analysis was performed by two-tailed Student's t-test for FTY720, and by one-way ANOVA followed by Dunnett's test for KSI-6666 (n = 5 rats /group; mean ± s.e.m.).c.Compound-induced change to respiratory function in mice.The test compound dissolved or suspended in 0.5% methyl cellulose 400 solution was orally administered to 8-week-old male BALB/cCr mice (Japan SLC).Two equimolar amount of hydrochloric acid (Wako) was mixed only in the suspension of KSI-6666.KSI-6666 was administered at 20 mg/kg and FTY720 at 5 mg/kg.Four hours after administration, spontaneously breathing awake mice unrestrained were placed in the whole-body plethysmographic chamber (Buxco) connected to a pressure transducer.Acclimatization of mice to the chamber was performed on the day before the assessment for 20 min and on the day of assessment for 17 min.Respiratory rate (f: breaths per minutes) and respiratory pressure curves (Penh) were recorded for 3 min by FinePointe Systems (Buxco).Statistical analysis was performed by either two-tailed Student's t-test or two-tailed Welch's t-test according to F-test results (n = 8 mice /group; mean ± s.e.m.).

d.
Representative histological images of the T cell transfer colitis model were exhibited (scale bar = 100 µm).
e. Efficacies of KSI-6666 (30 mg/kg/total) and KRP-203 (3 mg/kg/total) in oxazolone-induced colitis model.A total of 150 µL of 3% 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone; Sigma-Aldrich) dissolved in 100% ethanol (Wako) was applied on shaved back skins of 9-10-week-old male BALB/cCr mice for sensitization.One week after sensitization, 100 µL of 1% oxazolone dissolved in 50% ethanol oxazolone was intrarectally administered to the mice.On the same day, test compounds suspended in the vehicle consisting of a 1:9 mixture of 0.5% methyl cellulose 400 solution (Wako) and distilled water (Otsuka) were orally administered to mice twice (before and after sensitization).Mice were sacrificed 24 h after sensitization, and colons were collected and weighed.Homogenates of colons were prepared using TisseLyser (QIAGEN) and the amount of interleukin (IL)-4 protein was evaluated using a Mouse IL-4 Quantikine ELISA Kit (R&D Systems).Change in body weight was calculated by the difference between the day of intrarectal oxazolone administration and the end of study.a. Significant protein-ligand interactions of (R)-KSI-6666 relative to W146 in protein-ligand interaction fingerprint (PLIF) analysis.The interaction between the compound and S1PR1 during MetaD simulation was represented in a fingerprint scheme by the PLIF tool.The interaction between the compound and each amino acid residue of S1PR1 was evaluated in two main categories: potential-energy based contacts and surface contacts.When an interaction above a threshold was observed for the residue, a fingerprint bit representing the interaction was assigned.Hydrogen bonding, ionic interaction, and π interaction (arene attraction) were identified as potential contacts with energies >0.5 kcal/mol and assigned as fingerprint bits in this analysis.For hydrogen bonding, each amino acid residue was broken down into side chain (SC) and backbone (BB) categories, and each was classified as either electron donor or acceptor.
When a surface contact with area >15 Å 2 was observed, a fingerprint bit was assigned.The probability of each bit occurring for each residue for (R)-KSI-6666 compared with W146 was calculated as Qb, where Qb takes values from −1 to 1.A higher positive value indicates a higher probability of the bit occurring for (R)-KSI-6666.The columns show the value of Qb for each residue.The fingerprint bits shown are total surface contacts (left); and arene attraction (representing π interaction), ionic attraction, and BB hydrogen acceptor, BB hydrogen donor,

Chemistry
All reagents and solvents were purchased from commercial sources and used without further purification.All moisture and air sensitive reactions were carried out under an argon atmosphere.
Reactions were monitored by thin layer chromatography (TLC) using Merck Kieselgel 60 F254 plates.Flash column chromatography was performed on YAMAZEN amino packed inject column and Biotage prepacked columns using an automated flash chromatography system (Biotage, Isolera One). 1 H NMR spectra was recorded on a Bruker AV400 M spectrometer at 400.1 MHz.
Chemical shifts (δ) were reported in parts per million (ppm) units using tetramethylsilane as an internal standard.Data were presented as follows; chemical shift, integration, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), and coupling constant. 13C NMR spectra were recorded on a Bruker AV400M spectrometer at 100.6 MHz with complete proton decoupling.Chemical shifts (δ) were reported in parts per million (ppm) with the solvent as the internal reference (δ 77.16 in CDCl3).High resolution mass spectra (HRMS) was recorded on an Agilent Technologies 6520 Accurate-Mass Q-TOF instrument using electrospray ionization (ESI, positive or negative ion mode).
SC hydrogen acceptor, and SC hydrogen donor interactions with (R)-KSI-6666 (right, different interactions shown in different colors).b-d.Surface and cellular expression levels of HiBiT-tagged wild-type and mutant human S1PR1.HEK293 cells expressing HiBiT-tagged human S1PR1 (wild-type, or Val124 or Leu124 mutant) with mVenus were subjected to extracellular monitoring of HiBiT by using a Nano Glo HiBiT Extracellular Detection System (Promega) (b), or cellular monitoring of HiBiT by using a Nano Glo HiBiT Lytic Detection System (Promega) (c).The fluorescence intensity of mVenus, expressed as an internal control in each cell type, was evaluated (d).Data are the mean ± s.e.m. of three independent experiments.Source data are provided as a Source Data file.