ST-2191, an Anellated Bismorpholino Derivative of Oxy-Fingolimod, Shows Selective S1P1 Agonist and Functional Antagonist Potency In Vitro and In Vivo

Abstract

Sphingosine 1-phosphate (S1P) is an extensively studied signaling molecule that contributes to cell proliferation, survival, migration and other functions through binding to specific S1P receptors. The cycle of S1P₁ internalization upon S1P binding and recycling to the cell surface when local S1P concentrations are low drives T cell trafficking. S1P₁ modulators, such as fingolimod, disrupt this recycling by inducing persistent S1P₁ internalization and receptor degradation, which results in blocked egress of T cells from the secondary lymphoid tissues. The approval of these compounds for the treatment of multiple sclerosis has placed the development of S1PR modulators in the focus of pharmacological research, mostly for autoimmune indications. Here, we report on a novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P₁ agonist and functional antagonist potency. ST-2191 is also effective in reducing the lymphocyte number in mice, and this effect is not dependent on phosphorylation by sphingosine kinase 2 for activity. These data show that ST-2191 is a novel S1P₁ modulator, but further experiments are needed to analyze the therapeutic impact of ST-2191 in animal models of autoimmune diseases.

Keywords: S1P1 receptor; ST-2191; anellated bismorpholino; functional antagonism; lymphopenia; sphingosine 1-phosphate.

Figure 1

Scheme of the chemical synthesis route for ST-2191 starting from the oxy-analogue of fingolimod (O-FTY). The main product of this reaction is ST-2192 (30%), while ST-1894 (15%) (see also [18]) and ST-2191 (5%) were generated as side products. Synthesis details can be found in the Supplementary File. (a) 1,2-dibromoethane, acetone; (b) crystallization with acetone, DCM/ammonia; (c) extraction with DCM; (d) flash chromatography with DCM/methanol.

Figure 2

Two-dimensional molecular model of interactions between ST-2191 and amino acid residues of the binding pocket of the S1P₁ receptor. Molecular modelling experiments were performed as explained in the Materials and Methods section. Green colored circles represent hydrophobic amino acids, while pink circles represent polar amino acids. Red contour is drawn around the acidic and blue around basic amino acids. The blue clouds in the structure of ST-2191 indicate the surface exposed to the solvent, while the blue halo around some amino acids indicates an interaction with the ligand, where a larger halo means a stronger interaction. Hydrogen bonds are represented by green arrows.

Figure 3

S1P receptor activation profile of ST-2191. CHO cells overexpressing the S1P₁, S1P₂, S1P₃, S1P₄ or S1P₅ were stimulated for 10 min with vehicle (DMSO, ctrl), S1P (A,B), ST-2191 (A,B) or ST-2192 (B) in serum-free medium and were further processed for Western blot detection of phospho-p42/p44-MAPK and total p42/p44, as explained in the Materials and Methods section. Blots show one representative out of three independent experiments. (C) Bands of phospho-p42/p44-MAPK were quantified by using ImageJ software. Results are presented as means ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, **** p < 0.0001 compared to DMSO-treated control. A non-linear regression analysis was used to calculate the half-maximal effective concentration (EC₅₀) of ST-2191 for the S1P₁ (R² = 0.96) as 1.99 μM.

Figure 4

Prolonged effect of ST-2191 on S1P₁ signaling. Confluent and starved CHO-S1P₁ cells were stimulated with vehicle (DMSO, ₋), S1P or ST-2191 in a serum-free medium for 3 h (A) or 3 h with a wash-out period of 21 h (B) and were further stimulated with a new pulse of S1P for 10 min. Cells were lysed and processed for Western blot detection of phospho- and total-p42/p44-MAPK, as explained in the Materials and Methods section.

Figure 5

Prolonged effect of ST-2191 on S1P₁ internalization. Confluent and starved CHO-S1P₁ cells in 24-well plates were stimulated with DMSO (control), S1P or ST-2191 in a serum-free medium for 3 h (A) or 3 h with a wash-out period of 21 h (B). After fixation, S1P₁-ELISA was performed as explained in the Materials and Methods section. Results are expressed as % of control and are depicted as means ± S.D. (n = 3; *** p < 0.001, **** p < 0.0001).

Figure 6

ST-2191-induced decrease in lymphocyte numbers in mice. Wildtype mice (wt) or mice deficient in Sphk2 (Sphk2 ko) were injected in the peritoneum with a single dose of 1 mg/kg ST-2191 (in DMSO/PBS) or vehicle (DMSO in PBS), and after 24 h, blood was collected and lymphocyte number was measured as explained in the Materials and Methods section. Results are presented as means ± S.E.M. (n = 5 in each group) and significance was calculated by Mann–Whitney U test (** p < 0.01 compared to vehicle-treated wt mice; ^## p < 0.01 compared to vehicle-treated Sphk2 ko mice).