(S)-FTY720-Vinylphosphonate, an analogue of the immunosuppressive agent FTY720, is a pan-antagonist of sphingosine 1-phosphate GPCR signaling and inhibits autotaxin activity

Abstract

FTY720 (Fingolimod™), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P 1 receptor internalization and targeting to the proteasomal degradative pathway, and thus acts as a functional antagonist of S1P 1 by depleting the functional S1P 1 receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)-and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P 1 (EC 50 20 ± 3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P 1,3,4 (K i 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P 2 , and S1P 5 . Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K i values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)-and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P 2 transcripts and traces of S1P 5 . (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The biological effects of the (R)-and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets. NIH Public Access

FTY720 (Gilenya) Phosphate Selectivity of Sphingosine 1-Phosphate Receptor Subtype 1 (S1P1) G Protein-coupled Receptor Requires Motifs in Intracellular Loop 1 and Transmembrane Domain 2

“…Anti-FLAG M2 monoclonal antibody and cycloheximide were purchased from Sigma-Aldrich. FTY720PN was synthesized (25) and dissolved as described previously (21). [ 32 P]S1P was synthesized enzymatically using recombinant sphingosine kinase 1 as described previously (29); the specific activity was 86 cpm/fmol.…”

“…1F and Table 1). Effects of Mutations on S1P 2 Receptor Activation by FTY720P-FTY720P is a high affinity agonist of all the S1P receptors except S1P 2 , although high concentrations of FTY720P activate S1P 2 -mediated Ca 2ϩ mobilization in some experimental settings (21). In RH7777 cells co-transfected with G␣ q , FTY720P elicited a weak but measurable calcium efflux response from the WT S1P 2 receptor (EC 50 ϭ 359 Ϯ 69 nM, E max ϭ 56% of the maximal S1P response; Table 1).…”

“…Previously, we characterized FTY720PN against the S1P 1-5 receptors expressed in HTC4 stable cell lines and found that the FTY720PN was a strong activator of S1P 1 but did not activate S1P 2 (21). We tested activation of S1P 1 , S1P 2 , and the S1P 2 (IC1-TM2) S1P1 receptors for activation by FTY720PN in transiently transfected HTC4 cells (Fig.…”

“…The S1P 2 receptor is not activated by para-substituted aromatic ligands such as FTY720P (10,21). We have applied computational modeling-guided mutagenesis studies for mapping the common and distinguishing features of ligand recognition by endothelial differentiation gene family lysophosphatidic acid and S1P G protein-coupled receptors (22)(23)(24)(25)(26)(27).…”

“…Preferred agonists retain substantial flexibility when bound. Using the S1P 1 /S1P 2 chimera, we tested other S1P 1 -selective analogs including (R)-FTY720 phosphonate (FTY720PN) (21,28), which also activated this chimera. Interestingly, neither FTY720P nor FTY720PN caused the selective internal sequestration of the S1P 1 /S1P 2 chimera characteristic for S1P 1 , indicating that additional structural elements are required for the selective trafficking of the S1P 1 receptor to the degradative pathway by these types of ligands.…”