Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Abstract

The immunomodulatory drug fingolimod (FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). [1,2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor.[1] The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. [3] In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system [4] and on various tumor cell types. [5,6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, [7] oligodendrocyte differentiation, and cell survival [8,9] and neurogenesis. [10,11] To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, [12] we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC 50 = 270 nm), but has modest selectivity against S1P1 (EC 50 = 3140 nm) and S1P4 (EC 50 = 100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists.To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). [13] Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring,~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, o...

Galactosylsphingosine (psychosine)-induced demyelination is attenuated by sphingosine 1-phosphate signalling

“…Here, for the first time, we showed that psychosine can directly induce a concentration-dependent demyelination, as expressed by a decrease in the levels of MOG, PLP and MBP, and that pFTY720 can attenuate these effects. These findings are in agreement with our and other previous reports demonstrating that pFTY720 rescues myelination (Coelho et al, 2007;Jung et al, 2007;Mattes et al, 2010;Miron et al, 2008a,b;Sheridan and Dev, 2012). Interestingly, unlike LPC, psychosine treatment alone did not induce the release of IL6, TNFα or IL1β from cerebellar slices, which was in agreement with our astrocyte data.…”

S1P1 receptor subtype inhibits demyelination and regulates chemokine release in cerebellar slice cultures

“…These effects may be attributed to an enhanced survival of mature oligodendrocytes and/or stabilization of the myelin sheath. In previous studies we have shown that pFTY720 increases the number of MBP-positive mature oligodendrocytes (Mattes et al, 2010).…”

“…We have also previously demonstrated that S1PR activation increases the number of MBP-positive mature oligodendrocytes in culture (Mattes et al, 2010). Here, using organotypic cerebellar slices, we determine the role of S1P1Rs in demyelination.…”

Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond

“…However, there is currently no evidence to support this notion. Although some selective and orally active S1PR5 agonists have been synthesized 104 , their in vivo effects have not yet been reported.…”