Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis

Fingolimod (Gilenya) received regulatory approval from the US FDA in 2010 as the first-in-class sphingosine 1-phosphate (S1P) receptor (S1PR) modulator and was the first oral disease-modifying therapy (DMT) used for the treatment of the relapsing forms of multiple sclerosis (MS). Development of this new class of therapeutic compounds has continued to be a pharmacological goal of high interest in clinical trials for treatment of various autoimmune disorders, including MS. S1P is a physiologic signaling molecule that acts as a ligand for a group of cell surface receptors. S1PRs are expressed on various body tissues and regulate diverse physiological and pathological cellular responses involved in innate and adaptive immune, cardiovascular, and neurological functions. Subtype 1 of the S1PR (S1PR1) is expressed on the cell surface of lymphocytes, which are well known for their major role in MS pathogenesis and play an important regulatory role in the egress of lymphocytes from lymphoid organs to the lymphatic circulation. Thus, S1PR1-directed pharmacological interventions aim to modulate its role in immune cell trafficking through sequestration of autoreactive lymphocytes in the lymphoid organs to reduce their recirculation and subsequent infiltration into the central nervous system. Indeed, receptor subtype selectivity for S1PR1 is theoretically favored to minimize safety concerns related to interaction with other S1PR subtypes. Improved understanding of fingolimod's mechanism of action has provided strategies for the development of the more selective second-generation S1PR modulators. This selectivity serves to reduce the most important safety concern regarding cardiac-related side effects, such as bradycardia, which requires prolonged first-dose monitoring. It has led to the generation of smaller molecules with shorter half-lives, improved onset of action with no requirement for phosphorylation for activation, and preserved efficacy. The shorter half-lives of the second-generation agents allow for more rapid reversal of their pharmacological effects following treatment discontinuation. This may be beneficial in addressing further treatment-related complications in case of adverse events, managing serious or opportunistic infections such as progressive multifocal leukoencephalopathy, and eliminating the drug in pregnancies. In March 2019, a breakthrough in MS treatment was achieved with the FDA approval for the second S1PR modulator, siponimod (Mayzent), for both active secondary progressive MS and relapsing-remitting MS. This was the first oral DMT specifically approved for active forms of secondary progressive MS. Furthermore, ozanimod received FDA approval in March 2020 for treatment of relapsing forms of MS, followed by subsequent approvals from Health Canada and the European Commission. Other second-generation selective S1PR modulators that have been tested for MS, with statistically significant data from phase II and phase III clinical studies, include ponesimod (ACT-128800), ceralifimod (ONO-4641), and amiselimod (MT-1303). This review covers the available data about the mechanisms of action, pharmacodynamics and kinetics, efficacy, safety, and tolerability of the various S1PR modulators for patients with relapsing-remitting, secondary progressive, and, for fingolimod, primary progressive MS.

Cyclical and Dose-Dependent Responses of Adult Human Mature Oligodendrocytes to Fingolimod

What is the cardiovascular effect of S1P receptor modulator therapy in multiple sclerosis? Sphingosine 1-phosphate (S1P) receptor (S1PR) modulators are among the most efficient therapies for multiple sclerosis. As small molecules, they are not only acting on the immune but on cardiovascular and nervous systems as well. Short-term effects of S1PR modulators on the cardiovascular system have already been extensively described, while long-term effects are less known. Our review describes the mechanisms of action and the short- and long-term effects of these therapeutic agents on the cardiovascular system in different clinical trials. We systematically reviewed the literature that had been published by January 2022. One hundred seven articles were initially identified by title and abstract using targeted keywords, and thirty-nine articles with relevance to cardiovascular effects of S1PR therapy in multiple sclerosis patients were thereafter considered, including their references for further accurate clarification. Studies on fingolimod, the first S1PR modulator approved for treating multiple sclerosis, primarily support the safety profile of this therapeutic class. The second-generation therapeutic agents along with a different treatment initiation approach helped mitigate several of the cardiovascular adverse effects that had previously been observed at the start of treatment. The heart rate may decrease when initiating S1PR modulators and, less commonly, the atrioventricular conduction may be prolonged, requiring cardiac monitoring for the first 6 h of medication. Continuous therapy with S1PR modulators can increase blood pressure values; therefore, the presence of arterial hypertension should be checked during long-term treatment. Periodic surveillance of the cardiovascular and autonomic functions can help predict cardiac outcomes and prevent possible adverse events in S1PR modulators treatment. Further studies with longer follow-ups are needed, especially for the second-generation of S1PR modulators, to confirm the safety profile of this therapeutic class.

FTY720 (Fingolimod) is a first-in-class sphingosine 1-phosphate (S1P) receptor modulator that inhibits S1P-dependent lymphocyte egress from secondary lymphoid organs. Oral administration of FTY720 shows a superior efficacy compared to interferon (IFN)-β in relapsing remitting multiple sclerosis (MS), the most common inflammatory disorder of the central nervous system (CNS) and in experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-dependent animal model for MS. FTY720 is a structural analogue of sphingosine and is rapidly converted to the (S)-enantiomer of FTY720-phosphate [ (S)-FTY720-P)] by sphingosine kinases. (S)-FTY720-P binds 4 types of S1P receptors (S1P1, S1P3, S1P4, and S1P5), induces internalization and degradation of lymphocytic S1P1, and inhibits S1P responsiveness of lymphocytes. Consequently, (S)-FTY720-P acts as a functional antagonist at S1P1 thereby inhibiting S1P-S1P1 axis-mediated lymphocyte egress from secondary lymphoid organs. Based on this mechanism, FTY720 sequesters myelin antigen-specific CD4 T cells including interleukin 17-expressing helper T cells (Th17 cells) and IFN-γ-expressing type 1 helper T cells (Th1 cells) into the lymph nodes, and reduces the infiltration of these Th cells into the CNS in mouse EAE. Since FTY720 was approved by the United States Food and Drug Administration in September 2010 as a first-line treatment for relapsing remitting MS, it is presumed that oral FTY720 provides a new therapeutic approach for MS.

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

FTY720 (Fingolimod) is the first of a new immunomodulator class: sphingosine 1-phosphate (S1P) receptor modulator. We have found FTY720 by chemical modification of a natural product, myriocin derived from Isaria sinclairii, a kind of vegetative wasp. FTY720 is orally active and is highly effective in various autoimmune disease models including experimental autoimmune encephalomyelitis (EAE), adjuvant- or collagen-induced arthritis, and lupus nephritis. Particularly, oral administration of FTY720 shows marked therapeutic effects on EAE in mice with a significant reduction of demyelination and T cell infiltration in the central nervous system. A most striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating effects. It is revealed that the reduction of circulating lymphocytes by FTY720 is due to sequestration of lymphocytes into secondary lymphoid organs. FTY720 is rapidly converted to FTY720-phosphate (FTY720-P) by sphingosine kinases. FTY720-P acts as a potent agonist at S1P receptor type 1 (S1P1), internalizes S1P1 on lymphocytes, and inhibits migration of lymphocytes toward S1P. It is highly likely that immunomodulating effects of FTY720 are caused by inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs. Moreover, it is suggested that direct effects of FTY720-P on neural cells via S1P receptors promote neuroprotection. Since FTY720 possesses a novel mechanism of action and is highly effective in relapsing remitting multiple sclerosis patients, it is presumed that oral FTY720 provides a new therapeutic approach for autoimmune diseases including multiple sclerosis.

Until recently, all approved multiple sclerosis (MS) disease treatments were administered parenterally. Oral fingolimod was approved in September 2010 by the US Food and Drug Administration to reduce relapses and disability progression in relapsing forms of MS. In the clinical trials that led to approval, fingolimod reduced not only acute relapses and magnetic resonance imaging lesion activity but also disability progression and brain volume loss, suggesting preservation of tissue. Fingolimod's mechanism of action in MS is not known with certainty. Its active form, fingolimod-phosphate (fingolimod-P), is a sphingosine 1-phosphate receptor (S1PR) modulator that inhibits egress of lymphocytes from lymph nodes and their recirculation, potentially reducing trafficking of pathogenic cells into the central nervous system (CNS). Fingolimod also readily penetrates the CNS, and fingolimod-P formed in situ may have direct effects on neural cells. Fingolimod potently inhibits the MS animal model, experimental autoimmune encephalomyelitis, but is ineffective in mice with selective deficiency of the S1P₁ S1PR subtype on astrocytes despite normal expression in the immune compartment. These findings suggest that S1PR modulation by fingolimod in both the immune system and CNS, producing a combination of beneficial anti-inflammatory and possibly neuroprotective/reparative effects, may contribute to its efficacy in MS. In clinical trials, fingolimod was generally safe and well tolerated. Its interaction with S1PRs in a variety of tissues largely accounts for the reported adverse effects, which were seen more frequently with doses 2.5 to 10x the approved 0.5 mg dose. Fingolimod's unique mechanism of action distinguishes it from all other currently approved MS therapies.

Fingolimod: Direct CNS effects of sphingosine 1-phosphate (S1P) receptor modulation and implications in multiple sclerosis therapy

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator, binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), and is approved in multiple countries for treating adults with relapsing forms of multiple sclerosis (MS) or moderately to severely active ulcerative colitis (UC). Other S1P receptor modulators have been approved for the treatment of MS or are in clinical development for MS or UC, but it is unknown whether these compounds bind competitively with each other to S1P1 or S1P5. We developed a competitive radioligand binding assay using tritiated ozanimod and demonstrate full displacement of ozanimod by S1P (endogenous ligand), suggesting that ozanimod binds to the S1P1 and S1P5 orthosteric binding sites. S1P receptor modulators FTY720-p, siponimod, etrasimod, ponesimod, KRP-203-p, and amiselimod-p also completely displacing radiolabeled ozanimod; thus, on a macroscopic level, all bind to the same site. Molecular docking studies support these results and predict the binding of each molecule to the orthosteric site of the receptors, creating similar interactions within S1P1 and S1P5. The absolute free energy perturbation method further validated key proposed binding modes. Functional potency tightly aligned with binding affinities across S1P1 and S1P5 and all compounds elicited S1P1-mediated β-arrestin recruitment. Since all the S1P modulators included in this study display similar receptor pharmacology and compete for binding at the same site, they can be considered interchangeable with one another. The choice of any one particular agent should therefore be made on the basis of overall therapeutic profile, and patients can be offered the opportunity to switch S1P medications without the potential concern of additive S1P pharmacology.

Cryo-EM in combination with biochemistry, flow cytometry and fluorescence microscopy reveals the activation of S1PR1 by CD69, a type II membrane protein, in cis to induce receptor internalization and thereby disrupt cellular responsiveness to S1P gradients.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) characterized by neuroinflammation, demyelination, and neurodegeneration. Among disease-modifying therapies, sphingosine 1-phosphate (S1P) receptor (S1PR) modulators such as fingolimod, also known as FTY720, have been shown to exert therapeutic effects through direct CNS actions at S1PRs (e.g. S1P1) expressed by astrocytes, beyond the originally proposed mechanism action of lymphocyte sequestration. This review highlights the emerging evidence linking S1P signaling to the vitamin B12 pathway, including transcobalamin 2 (TCN2) and CD320. Functional interaction between S1P1 signaling and CD320 expression was discovered by examining gene expression changes in immediate-early astrocytes (ieAstrocytes), the primary CNS cell type activated in response to neuroinflammatory stimuli. This discovery led to the identification of the physical interaction between fingolimod/sphingosine and TCN2 and the potentiation of CD320 internalization by this complex. These findings underscore the importance of CNS vitamin B12 levels in MS and likely other neurological diseases and help to explain the long-appreciated shared neurological symptoms between vitamin B12 deficiency and MS. Future research should investigate therapeutic strategies targeting the crosstalk between the sphingolipid and vitamin B12 pathways to enhance CNS vitamin B12 availability, which can promote neuroprotection in MS and related diseases.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds. https://doi.org/10.7554/eLife.33285.001 Introduction Pain is a complex sensation. It serves to protect organisms from harmful stimuli, but can also become chronic and debilitating following tissue injury and disease. Distinct cells and molecules detect noxious thermal and mechanical stimuli. Thermal pain is detected by thermosensitive TRP channels in subsets of nociceptors (Caterina et al., 2000; Vriens et al., 2011), and gentle touch is detected by Piezo2 channels in low-threshold mechanoreceptors (LTMRs) (Ranade et al., 2014; Woo et al., 2014). Aδ high-threshold mechanoreceptors (HTMRs) have been shown to play a key role in responses to painful mechanical stimuli (Arcourt et al., 2017; Ghitani et al., 2017). Recent studies have shown that there are at least two populations of HTMRs that mediate responses to noxious mechanical stimuli. The Npy2r+ subpopulation of HTMRs mediates fast paw withdrawal responses to pinprick stimulation and terminates as free nerve endings in the epidermis (Arcourt et al., 2017). The Calca+ subpopulation of circumferential-HTMRs responds to noxious force and hair pulling, and terminates as circumferential endings wrapped around guard hair follicles (Ghitani et al., 2017). Additionally, somatostatin-expressing interneurons of laminae I-III in the dorsal horn of the spinal cord receive input from nociceptors and are required for behavioral responses to painful mechanical stimuli (Duan et al., 2014). Despite these advances in defining the cells and circuits of mechanical pain, little is known about the molecular signaling pathways in mechanonociceptors. Here, we show that sphingosine 1-phosphate (S1P) is required for mechanical pain sensation. S1P is a bioactive lipid that signals via 5 G-protein coupled S1P Receptors (S1PRs 1–5). S1P signaling, mainly via S1PR1, plays a well-known role in immune cell migration and maturation (Spiegel and Milstien, 2003; Matloubian et al., 2004; Schwab et al., 2005). Additionally, recent studies have shown that S1PRs are expressed throughout the nervous system (Janes et al., 2014; Mair et al., 2011; Camprubí-Robles et al., 2013) and S1P signaling is associated with a variety of neuroinflammatory disorders, including multiple sclerosis (Brinkmann et al., 2010) and Alzheimer's disease (Couttas et al., 2014). S1P has been implicated in spontaneous pain (Camprubí-Robles et al., 2013) and thermal pain hypersensitivity (Mair et al., 2011; Finley et al., 2013; Weth et al., 2015), but due to conflicting accounts of S1P receptor expression in the CNS (Janes et al., 2014; Weth-Malsch et al., 2016) and PNS (Mair et al., 2011; Camprubí-Robles et al., 2013; Usoskin et al., 2015) as well as inconsistent reports on the effects of S1P on neuronal excitability (Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015) and pain behaviors (Mair et al., 2011; Camprubí-Robles et al., 2013; Finley et al., 2013; Weth et al., 2015), the role of S1P in somatosensation remains controversial. We found that mice lacking the S1P receptor S1PR3 display striking and selective deficits in behavioral responses to noxious mechanical stimuli. Likewise, peripheral blockade of S1PR3 signaling or S1P production impairs mechanical sensitivity. We show that S1P constitutively enhances the excitability of A mechanonociceptors (AMs) via closure of KCNQ2/3 potassium channels to tune mechanical pain sensitivity. The effects of S1P are completely dependent on S1PR3. While previous studies have shown that elevated S1P triggers acute pain and injury-evoked thermal sensitization (Mair et al., 2011; Camprubí-Robles et al., 2013), we now demonstrate that baseline levels of S1P are necessary and sufficient for setting normal mechanical pain thresholds. By contrast, elevated S1P selectively triggers thermal sensitization via activation of TRPV1+ heat nociceptors, with no effect on mechanical hypersensitivity. Our findings uncover an essential role for constitutive S1P signaling in mechanical pain. Results To identify candidate genes underlying mechanosensation, we previously performed transcriptome analysis of the sensory ganglia innervating the ultra-sensitive tactile organ (the star) of the star-nosed mole (Gerhold et al., 2013). Immunostaining revealed the tactile organ is preferentially innervated by myelinated Aδ fibers (Gerhold et al., 2013), which are primarily mechanosensitive. While our original analysis focused on ion channels enriched in the neurons of the star organ, our dataset also revealed enrichment of several components of the S1P pathway, including S1pr3. Likewise, single-cell RNA seq of mouse dorsal root ganglion (DRG) neurons revealed S1pr3 expression in a subset of myelinated mechanoreceptors (Usoskin et al., 2015) in addition to a subpopulation of peptidergic C nociceptors. S1P promotes excitability in small-diameter, capsaicin-sensitive nociceptors (Mair et al., 2011; Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015). In addition, S1PR3 has been shown to mediate spontaneous pain triggered by elevated S1P and thermal sensitization following sterile tissue injury (Camprubí-Robles et al., 2013). However, no studies have examined the role of S1PR3 in mechanosensation or in regulating somatosensory behaviors under normal conditions. Given the enrichment of S1pr3 in mechanosensory neurons of the star-nosed mole and mouse, we hypothesized that S1P signaling via S1PR3 may also play a role in mechanosensation. Thus, we set out to define the role of S1P signaling and S1PR3 in somatosensory mechanoreceptors. S1PR3 mediates acute mechanical pain We first examined a variety of somatosensory behaviors in mice lacking S1PR3 (Kono et al., 2004) (S1pr3tm1Rlp/Mmnc; referred to herein as S1PR3 KO). We initially investigated baseline responses to mechanical stimuli. S1PR3 KO mice displayed a dramatic loss of mechanical sensitivity (Figure 1A; see Figure 1—source data 1), as von Frey paw withdrawal thresholds were significantly elevated in S1PR3 KO mice relative to WT and S1PR3 HET littermates (mean thresholds: 1.737 g vs. 0.736 and 0.610 g, respectively). Moreover, S1PR3 KO mice demonstrated decreased responses to a range of noxious tactile stimuli (2–6 g; Figure 1B) and to noxious pinprick stimulation (Figure 1C), but normal responsiveness to innocuous tactile stimuli (0.6–1.4 g; Figure 1B). S1PR3 KO mice exhibited normal tape removal attempts (Ranade et al., 2014) (Figure 1D), righting reflexes (Figure 1—figure supplement 1A), radiant heat withdrawal latencies (Figure 1E), and itch-evoked scratching (Figure 1—figure supplement 1B). These results demonstrate a selective role for S1PR3 in acute mechanical pain. Figure 1 with 1 supplement see all Download asset Open asset S1PR3 mediates acute mechanical pain. (A) von Frey 50% withdrawal threshold measurements for S1pr3+/+ (WT, N = 8), S1pr3+/- (HET, N = 7) and S1pr3-/- (KO, N = 12) mice. p<0.0001 (one-way ANOVA). Tukey-Kramer post hoc comparisons for KO and HET to WT indicated on graph. (B) von Frey force-response graph for WT (N = 8) versus KO (N = 12) animals; pgenotype <0.0001 (two-way ANOVA). Tukey HSD comparisons between genotypes are indicated for given forces. (C) % withdrawal to pinprick stimulation of hindpaw for HET versus KO animals; p<0.0001 (unpaired t-test; N = 5–7 mice per group). (D) Number of attempted removal bouts in tape assay for WT (N = 2), HET (N = 2), and KO (N = 5) mice; p=0.172 (one-way ANOVA). (E) Baseline radiant heat measurements for WT (N = 8), HET (N = 3), and KO (N = 5) mice; p=0.444 (one-way ANOVA). (F) von Frey 50% withdrawal threshold measurements for mice pre- and post-injection of 500 µM TY 52156 (N = 10), 10 µM W146 (N = 6), or 1% DMSO-PBS vehicle (N = 17); p=0.016, 0.650 (two-tailed paired t-test comparing vehicle- vs. drug-injected paw). (G) von Frey force-response graph for mice injected with either 1% DMSO-PBS (N = 4) or 500 µM TY 52156 (N = 4); ptreatment <0.0001 (two-way ANOVA). Tukey HSD comparisons were made between treatment groups and significant differences at a given force are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.002 Figure 1—source data 1 S1PR3 mediates acute mechanical pain. Related to Figure 1. https://doi.org/10.7554/eLife.33285.004 Download elife-33285-fig1-data1-v2.xlsx As a complement to our analysis of somatosensation in S1PR3 KO animals, we employed a pharmacological approach, using the S1PR3-selective antagonist TY 52156 (TY) (Nussbaum et al., 2015). Similar to the phenotype of knockout animals, intradermal injection of 500 µM TY into the mouse hindpaw (the site of testing) triggered a rapid and significant elevation in von Frey paw withdrawal thresholds (Figure 1F) and decreased responsiveness to noxious (2–6 g), but not innocuous (0.6–1.4 g), tactile stimuli (Figure 1G), without affecting noxious heat sensitivity (Figure 1—figure supplement 1C). By contrast, blockade of S1PR1 with the selective antagonist W146 (Finley et al., 2013) had no effect on baseline mechanical or thermal thresholds (Figure 1F; Figure 1—figure supplement 1C). Overall, these data show that S1PR3 signaling sets mechanical pain sensitivity. Endogenous S1P mediates acute mechanical pain We next asked whether peripheral S1P was required for the S1PR3-dependent effects on mechanosensation. We decreased S1P levels via injection of the sphingosine kinase inhibitor SKI II to block local production of S1P (Chiba et al., 2010) or elevated S1P levels via intradermal injection of S1P and measured behaviors 30 min after injection. Decreasing local S1P levels with SKI II significantly reduced mechanical sensitivity (Figure 2A; see Figure 2—source data 1), comparable to the hyposensitivity phenotype observed in S1PR3 KO mice (Figure 1A). Again, similar to what was observed in S1PR3 KO animals (Figure 1E), peripheral blockade of S1P production had no effect on baseline thermal sensitivity (Figure 1—figure supplement 1C). Surprisingly, injecting exogenous S1P (10 µM; maximum solubility in saline vehicle) had no effect on mechanical sensitivity (Figure 2A–B). However, as previously reported (Mair et al., 2011; Camprubí-Robles et al., 2013), S1P injection triggered S1PR3-dependent thermal hypersensitivity and spontaneous pain (Figure 2C–D), demonstrating that the lack of effect on mechanical hypersensitivity is not due to problems with S1P delivery or degradation. Figure 2 Download asset Open asset Endogenous S1P mediates acute mechanical pain. (A) von Frey 50% withdrawal measurements for mice pre- and post-injection of 50 µM SKI II (N = 8) or 10 µM S1P (N = 7); p=0.003, 0.604 (two-tailed paired t-tests). (B) von Frey force-response graph for animals injected with 10 µM S1P or 0.1% MeOH-PBS; pgenotype >0.05 (two-way ANOVA; N = 8 mice per group). No Tukey HSD comparisons at any force between genotypes were significant. (C) Intradermal cheek injection of 10 µM S1P, 2 µM, 0.2 µM, and 20 µL 0.3% methanol PBS (vehicle), with quantification of number of forepaw wipes over the 5 min post-injection interval; p<0.0001 (one-way ANOVA; N = 3 mice per condition). Dunnett's multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (D) Radiant heat normalized paw withdrawal latencies 20–30 min post injection of 15 µL 10 µM S1P, 0.2 µM S1P. or 0.3% methanol-PBS vehicle (i.d.) into the hind paw of S1PR3 WT or KO mice; p=0.0129 (one-way ANOVA; N = 3–10 mice per condition). Dunnett's multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (E) von Frey 50% withdrawal measurements for mice pre- (baseline) and post-injection of 50 µM SKI II (N = 14) and 0 (N = 4), 10 (N = 3), 75 (N = 4), or 200 nM S1P (N = 3; one-way ANOVA; p=0.0001). Tukey Kramer comparisons are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.005 Figure 2—source data 1 Endogenous S1P mediates acute mechanical pain. Related to Figure 2. https://doi.org/10.7554/eLife.33285.006 Download elife-33285-fig2-data1-v2.xlsx These data support a model whereby S1P constitutively activates S1PR3 to set normal mechanical pain thresholds. To further test this model, we asked if the mechanical hyposensitivity elicited after endogenous S1P depletion (via SKI II) could be rescued by local injection of exogenous S1P. Indeed, we found that injection of exogenous S1P reversed SKI II-induced mechanical hyposensitivity in a dose-dependent manner, and observed a maximal effect with 200 nM S1P (Figure 2E). Although quantification of native S1P levels in skin is inaccurate owing to avid lyase activity (Shaner et al., 2009), our data establish that baseline S1P levels are sufficient to maximally exert their effect on S1PR3-dependent mechanical pain, such that increased S1P does not evoke mechanical hypersensitivity, but diminished S1P leads to mechanical hyposensitivity. These data show that constitutive activation of S1PR3 by S1P is required for normal mechanosensitivity. S1PR3 is expressed in A mechanonociceptors and thermal nociceptors Our behavioral data showing distinct roles for S1PR3 in mechanonociception and thermal hypersensitivity suggest that S1PR3 is expressed in distinct subsets of somatosensory neurons. While a previous study suggested that all somatosensory neurons express S1PR3 (Camprubí-Robles et al., 2013), single cell RNA seq data suggests S1pr3 is not expressed by all DRG neurons (Usoskin et al., 2015), and no studies have performed quantitative analysis of S1PR3 staining or co-staining to define subpopulations of S1PR3+ neurons. We thus set out to characterize the somatosensory neuron subtypes expressing S1pr3 using in situ hybridization (ISH) of wild-type somatosensory ganglia and immunohistochemistry (IHC) in an S1pr3mCherry/+ reporter mouse (Sanna et al., 2016). We first used in situ hybridization (ISH) with a specific S1pr3 probe to examine expression patterns of S1pr3 (Figure 3A–B; see Supplementary file 1). In our experiments, 43% of cells from wild-type DRG expressed S1pr3. Co-ISH revealed that one population of S1pr3+ neurons represents Aδ mechanonociceptors (AMs). These cells expressed Scn1a (39.9% of all S1pr3+), a gene that encodes the Nav1.1 sodium channel, which mediates mechanical pain in Aδ fibers (Osteen et al., 2016). S1pr3+ cells also co-expressed Npy2r (20.4% of all S1pr3+), a marker of a subset of mechanonociceptive A fibers (Arcourt et al., 2017). S1pr3 was expressed in 70.6% of Scn1a+ cells and 72% of Npy2r+ cells, comprising a majority of both of these populations. Interestingly, a subset of cells co-expressed S1pr3 and the mechanically sensitive channel Piezo2, which is expressed by Aβ, Aδ, and C fibers (Ranade et al., 2014). The remaining S1pr3+ cells were Trpv1+ and/or Trpa1+ C nociceptors (67.1% of all S1pr3+), which are reported to overlap minimally with the Scn1a+ and Npy2r+ populations (Arcourt et al., 2017; Osteen et al., 2016). Figure 3 with 1 supplement see all Download asset Open asset S1pr3 is expressed in A mechanonociceptors and C thermal nociceptors. (A) (Top) Representative co-ISH of S1pr3 (green; left) with Scn1a, Npy2r, Piezo2, and Trpv1 (magenta; center) in sectioned DRG. Right column: overlay with co-localized regions colored white (10x air objective; scale = 100 µm). (B) Bar chart showing the % of total cells expressing the indicated marker (grey) and the % of total cells co-expressing both marker and S1pr3 (green). See Table S1 for quantification. (C) Representative IHC images of sectioned DRG from S1pr3mCherry/+ animals stained with anti-DsRed (green, S1PR3) and anti-Peripherin (left, magenta) or anti-NF200 (right, magenta). Arrows indicate co-stained cells. Images were acquired using a 10x air objective (scale = 100 µm). (D) Whole-mount skin IHC confocal images with anti-DsRed antibody (S1PR3, green) and anti-NefH antibody (NF200, magenta) in an S1pr3mCherry/+ animal (20x water objective; scale = 50 µm). Arrows indicate co-positive free nerves (left image). Arrowheads indicate NF200- free nerves (left) or S1PR3- circumferential fibers (right image). (E) Sectioned skin IHC with anti-DsRed (S1PR3) and anti-NefH (NF200, left, top right) or anti-DsRed (S1PR3) and anti-beta-tubulin III (BTIII, bottom right) antibody (magenta) in S1pr3mCherry/+ skin (20x air objective; scale = 50 µm). Arrows indicate co-positive free nerve endings (left), S1PR3-negative lanceolate/circumferential hair follicle endings (top right, arrow = circumferential, arrowhead = lanceolate), or S1PR3-negative putative Merkel afferent (bottom right). (F) (Left) Quantification of sectioned DRG IHC experiments showing % of S1PR3+ cells that co-stained with indicated markers (n > 250 cells per marker). (Right) Quantification of sectioned skin IHC experiments showing % of fibers positive for indicated marker that co-stained with S1PR3 (anti-DsRed; n = 10 images per marker from two animals). https://doi.org/10.7554/eLife.33285.007 We next used an S1pr3mCherry/+ reporter mouse, which produces a functional S1PR3-mCherry fusion protein (Sanna et al., 2016), as an independent strategy to explore S1PR3 expression and localization. This strategy was used because we found that anti-S1PR3 antibodies showed broad immunoreactivity in DRG from mice lacking S1PR3, and so we instead used anti-DsRed antibodies to probe expression of the S1PR3 fusion protein (Figure 3—figure supplement 1E). We found that 42.4% of S1PR3+ cells co-stained with anti-Peripherin, demonstrating that S1PR3 is expressed in a subset of small-diameter neurons. We also observed that 69.5% of S1PR3+ cells co-stained with anti-NF200, which marks medium and large-diameter myelinated neurons. Furthermore, we observed that S1PR3+ cells were primarily of small to medium diameter (11.3–35.1 µm), whereas all cells in the DRG ranged from 11.3 to 53.9 µm. Overall, these data support the expression of S1PR3 in subsets of small-diameter thermal nociceptors and medium-diameter mechanonociceptors (Figure 3F). Additionally, no significant differences were observed between WT and S1PR3 KO DRG in number of Trpa1+, Trpv1+, Peripherin+, NF200+, or IB4+ cells (Figure 3—figure supplement 1B–C,F,G). The mean diameters of Trpv1+ neurons (Figure 3—figure supplement 1D, left), NF200+ neurons (Figure 3—figure supplement 1G), or all neurons (Figure 3—figure supplement 1D, right) in WT versus KO DRG were not significantly different, suggesting no loss of major sensory neuronal subtypes in the S1PR3 KO. We then visualized S1PR3 expression in nerve fibers that innervate the skin using anti-DsRed antibodies in whole-mount immunohistochemistry (IHC; Figure 3D). The reporter animals showed no specific antibody staining in epidermal or dermal cells (Figure 3—figure supplement 1I), and single-cell RNA seq of a diverse array of mouse epidermal and dermal cells corroborates this lack of expression (Joost et al., 2016). We observed overlap of S1PR3-expressing free nerve endings with NF200+ myelinated free nerves and NF200- putative C-fiber endings (Figure 3F), but did not observe expression of S1PR3 in NF200+ circumferential or lanceolate hair follicle receptors, or in putative Merkel afferents (Figure 3D–E). β-tubulin III, PGP9.5 (pan-neuronal markers), and NF200 staining in S1PR3 KO skin displayed patterns of epidermal and dermal innervation similar to WT skin, suggesting the phenotypes observed in the S1PR3 KO mice are not due to developmental loss of sensory neuronal innervation (pPGP9.5= 0.443 (n = 93, 38 fibers); pNefH = 0.405 (n = 61, 28 fibers); pBTIII = 0.353 (n = 104, 89 fibers); two-tailed t-tests based on average number of fibers per field of view). These results support expression of S1PR3 in subsets of myelinated A mechanonociceptors and unmyelinated C nociceptors that terminate as free nerve endings. S1P activates thermal nociceptors but not putative AMs Live imaging of cultured DRG neurons from adult reporter animals showed expression of S1PR3-mCherry fusion protein in 48.3% of neurons, mirroring our ISH and IHC results (Figure 4A). To examine the effects of S1P on A mechanonociceptors and C nociceptors, we performed ratiometric calcium imaging and electrophysiology on DRG cultures from reporter mice. Interestingly, only 56.1 ± 22.4% of mCherry-expressing neurons were activated by 1 µM S1P (Representative trace in Figure 4B; representative images in Figure 4C), which our dose-response showed to be the saturating concentration for calcium influx (Figure 4D; EC50 = 155 nM). neurons were also capsaicin-sensitive (n > sensory neurons from S1PR3 KO animals did not to S1P, as (Camprubí-Robles et al., 2013), exhibited responses that were not significantly from WT neurons (Figure 3—figure supplement The mean diameter of neurons was ± whereas the mean diameter of neurons was ± two-tailed We also performed cell experiments with studies (Mair et al., 2011; Zhang et al., 2006; Li et al., 2015), found that S1P in capsaicin-sensitive small diameter cells (Figure This that only the small-diameter, S1PR3+ putative nociceptors are by S1P. We next asked whether the S1PR3+ diameter neurons represent the mechanonociceptors observed by ISH (Figure To this we asked whether the a selective of nociceptors (Osteen et al., 2016), triggers calcium influx in S1PR3-expressing neurons. Indeed, we found that ± of neurons expressed (Figure with our staining showing expression of S1pr3 in nociceptors and the role of neurons in mechanical pain in (Osteen et al., 2016). Figure Download asset Open asset S1P activates thermal nociceptors but not mechanonociceptors. (A) (Left) Representative of in cultured adult DRG neurons from one S1pr3mCherry/+ (Right) Quantification of % of total cells expressing S1pr3 from DRG ISH and from DRG cultures (N = 2 animals (B) Representative from calcium imaging showing two neurons, one which to 1 µM S1P, 1 µM and and one which only to (C) (Left) calcium imaging (left) and after addition of 1 µM S1P in S1pr3 cultured mouse DRG neurons. Bar (Right) % of neurons that are to 1 µM S1P in ratiometric calcium imaging (n > cells from imaging from animals). (D) of mean neuronal calcium to of S1P. and (N = 2 animals). Error bars represent mean ± SD. for all S1P from which EC50 was S1P were also (E) trace of a single wild-type neuron in response to addition of 1 µM S1P and 1 µM with of to S1P and one of one also to Bar = 2 (F) (Left) calcium imaging after addition of 500 nM in S1pr3mCherry/+ neurons, which were used instead of adult DRG neurons because to without (Right) % of neurons that are (N = 1 total S1PR3 KCNQ2/3 channels to excitability We next the molecular by which S1P signaling in nociceptors may mechanical pain. We performed on the medium-diameter S1pr3mCherry/+ DRG neurons = ± which did not display calcium influx (Figure In these cells, 1 µM S1P did not (Figure supplement 1A; see Figure data or in the of injection (Figure supplement 1A; Figure However, S1P the threshold to in an S1PR3-dependent (Figure Figure supplement 1B). Figure 5 with 1 supplement see all Download asset Open asset S1PR3 KCNQ2/3 channels to experiments were performed in S1pr3mCherry/+ or DRG neurons. (A) (Left) of a single in cell and after S1P (Right) % in after S1P for S1pr3mCherry/+ (left, n = 7) and KO (right, n = 12) neurons = two-tailed paired t-tests). (B) % in input after S1P or vehicle two-tailed paired t-test; n = cells per group). (C) The is by The was by of the from the and at n = cells. Data were with a and were measured at the indicated to 20 following a was using the of the after to indicated indicated all bars represent mean ± (D) of a single neuron in cell comparing pre- and using indicated of a single neuron in cell with was in % in after indicated µM S1P, 3 µM or for S1pr3mCherry/+ medium-diameter one-way ANOVA; n = using at % in after indicated = 100 µM for S1pr3mCherry/+ medium-diameter two-tailed paired t-test; n = Figure data 1 S1PR3 KCNQ2/3 channels to Related to Figure Download We then set out to the by which S1PR3 activity neuronal excitability using studies showed that S1P capsaicin-sensitive nociceptors by sodium and potassium et al., 2006; Li et al., 2015). We found that S1P had no such effects on S1PR3+ medium-diameter cells (Figure supplement By contrast, S1P triggered a in input (Figure with the closure of potassium channels. analysis revealed that the by S1P was by potassium (Figure Additionally, S1P significantly reduced (Figure supplement 1F; Figure in an S1PR3-dependent (Figure supplement As in Aδ neurons are primarily mediated by KCNQ2/3 potassium channels et al., et al., we that S1P may modulation of these channels. Furthermore, the of the

Lessons from S1P receptor targeting in multiple sclerosis

Sphingosine 1-phosphate (S1P) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Lysophospholipid receptors [96]) are activated by the endogenous lipid sphingosine 1-phosphate (S1P). Originally cloned as orphan members of the endothelial differentiation gene (edg) family [16, 123], the receptors are currently designated as S1P1R through S1P5R [73, 16, 123]. Their gene nomenclature has been codified as human S1PR1, S1PR2, etc. (HUGO Gene Nomenclature Committee, HGNC) and S1pr1, S1pr2, etc. for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function. All S1P receptors (S1PRs) have been knocked-out in mice constitutively and in some cases, conditionally. S1PRs, particularly S1P1, are expressed throughout all mammalian organ systems. Ligand delivery occurs via two known carriers (or "chaperones"): albumin and HDL-bound apolipoprotein M (ApoM), the latter of which elicits biased agonist signaling by S1P1 in multiple cell types [18, 53]. The five S1PRs, two chaperones, and active cellular metabolism have complicated analyses of receptor ligand binding in native systems. Signaling pathways and physiological roles have been characterized through radioligand binding in heterologous expression systems, targeted deletion of the different S1PRs, and most recently, mouse models that report in vivo S1P1R activation [101, 103]. The structures of S1P1 [180, 69, 108, 184], S1P2 [32], S1P3[116, 187], and S1P5 [110, 185] are solved, and confirmed aspects of ligand binding, specificity, and receptor activation, determined previously through biochemical and genetic studies [69, 17]. fingolimod (FTY720), the first FDA-approved drug to target any of the lysophospholipid receptors, binds as a phosphorylated metabolite to four of the five S1PRs, and was the first oral therapy for multiple sclerosis (MS) [35]. Second-generation S1PR modulators siponimod, ozanimod, and ponesimod that target S1P1 and S1P5 are also FDA approved for the treatment of various MS forms [16, 123]. In 2021, ozanimod became the first S1PR modulator to be FDA approved for the treatment of ulcerative colitis [145]. The mechanisms of action of fingolimod and other S1PR-modulating drugs now in development include binding S1PRs in multiple organ systems, e.g., immune and nervous systems, although the precise nature of their receptor interactions requires clarification [141, 37, 63, 64].

Ponesimod is a sphingosine 1-phosphate (S1P) receptor (S1PR) modulator that was recently approved for treating relapsing forms of multiple sclerosis (MS). Three other FDA-approved S1PR modulators for MS – fingolimod, siponimod, and ozanimod – share peripheral immunological effects via common S1P1 interactions, yet ponesimod may access distinct central nervous system (CNS) mechanisms through its selectivity for the S1P1 receptor. Here, ponesimod was examined for S1PR internalization and binding, human astrocyte signaling and single-cell RNA-seq (scRNA-seq) gene expression, and in vivo using murine cuprizone-mediated demyelination. Studies confirmed ponesimod’s selectivity for S1P1 without comparable engagement to the other S1PR subtypes (S1P2,3,4,5). Ponesimod showed pharmacological properties of acute agonism followed by chronic functional antagonism of S1P1. A major locus of S1P1 expression in the CNS is on astrocytes, and scRNA-seq of primary human astrocytes exposed to ponesimod identified a gene ontology relationship of reduced neuroinflammation and reduction in known astrocyte disease-related genes including those of immediate early astrocytes (ieAstrocytes) that have been strongly associated with disease progression in MS animal models. Remarkably, ponesimod prevented cuprizone-induced demyelination selectively in the cingulum, but not in the corpus callosum. These data support the CNS activities of ponesimod through S1P1, including protective, and likely selective, effects against demyelination in a major connection pathway of the brain, the limbic fibers of the cingulum, lesions of which have been associated with several neurologic impairments including MS fatigue.