Imidazole-based sphingosine-1-phosphate transporter Spns2 inhibitors.

Sphingosine-1-phosphate (S1P) is a chemotactic lipid that influences immune cell positioning. S1P concentration gradients are necessary for proper egress of lymphocytes from the thymus and secondary lymphoid tissues. This trafficking is interdicted by S1P receptor modulators, and it is expected that S1P transporter (Spns2) inhibitors, by reshaping S1P concentration gradients, will do the same. We previously reported SLF1081851 as a prototype Spns2 inhibitor, which provided a scaffold to investigate the importance of the oxadiazole core and the terminal amine. In this report, we disclose a structure-activity relationship study by incorporating imidazole as both a linker and surrogate for a positive charge in SLF1081851. In vitro inhibition of Spns2-dependent S1P transport in HeLa cells identified 7b as an inhibitor with an IC50 of 1.4 ± 0.3 µM. The SAR studies reported herein indicate that imidazolium can be a substitute for the terminal amine in SLF1081851 and that Spns2 inhibition is highly dependent on the lipid alkyl tail length.

Lipophilic tail modifications of 2-(hydroxymethyl)pyrrolidine scaffold reveal dual sphingosine kinase 1 and 2 inhibitors.

The sphingosine 1-phosphate (S1P) signaling pathway is an attractive target for pharmacological manipulation due to its involvement in cancer progression and immune cell chemotaxis. The synthesis of S1P is catalyzed by the action of sphingosine kinase 1 or 2 (SphK1 or SphK2) on sphingosine and ATP. While potent and selective inhibitors of SphK1 or SphK2 have been reported, development of potent dual SphK1/SphK2 inhibitors are still needed. Towards this end, we report the structure-activity relationship profiling of 2-(hydroxymethyl)pyrrolidine-based inhibitors with 22d being the most potent dual SphK1/SphK2 inhibitor (SphK1 Ki = 0.679 µM, SphK2 Ki = 0.951 µM) reported in this series. 22d inhibited the growth of engineered Saccharomyces cerevisiae and decreased S1P levels in histiocytic lymphoma myeloid cell line (U937 cells), demonstrating inhibition of SphK1 and 2 in vitro. Molecular modeling studies of 22d docked inside the Sph binding pocket of both SphK1 and SphK2 indicate essential hydrogen bond between the 2-(hydroxymethyl)pyrrolidine head to interact with aspartic acid and serine residues near the ATP binding pocket, which provide the basis for dual inhibition. In addition, the dodecyl tail adopts a "J-shape" conformation found in crystal structure of sphingosine bound to SphK1. Collectively, these studies provide insight into the intermolecular interactions in the SphK1 and 2 active sites to achieve maximal dual inhibitory activity.

Mechanism of sphingosine 1-phosphate clearance from blood.

The interplay of sphingosine 1-phosphate (S1P) synthetic and degradative enzymes as well as S1P exporters creates concentration gradients that are a fundamental to S1P biology. Extracellular S1P levels, such as in blood and lymph, are high relative to cellular S1P. The blood-tissue S1P gradient maintains endothelial integrity while local S1P gradients influence immune cell positioning. Indeed, the importance of S1P gradients was recognized initially when the mechanism of action of an S1P receptor agonist used as a medicine for multiple sclerosis was revealed to be inhibition of T-lymphocytes' recognition of the high S1P in efferent lymph. Furthermore, the increase in erythrocyte S1P in response to hypoxia influences oxygen delivery during high altitude acclimatization. However, understanding of how S1P gradients are maintained is incomplete. For example, S1P is synthesized but is only slowly metabolized by blood yet circulating S1P turns over quickly by an unknown mechanism. Prompted by the counterintuitive observation that blood S1P increases markedly in response to inhibition S1P synthesis (by sphingosine kinase 2 (SphK2)), we studied mice wherein several tissues were made deficient in either SphK2 or S1P degrading enzymes. Our data reveal a mechanism whereby S1P is de-phosphorylated at the hepatocyte surface and the resulting sphingosine is sequestered by SphK phosphorylation and in turn degraded by intracellular S1P lyase. Thus, we identify the liver as the primary site of blood S1P clearance and provide an explanation for the role of SphK2 in this process. Our discovery suggests a general mechanism whereby S1P gradients are shaped.

Sphingosine Kinase 2 Deficiency Attenuates Kidney Fibrosis via IFN-γ.

Maladaptive repair after AKI may lead to progressive fibrosis and decline in kidney function. Sphingosine 1-phosphate has an important role in kidney injury and pleiotropic effects in fibrosis. We investigated the involvement of sphingosine kinase 1 and 2 (SphK1 and SphK2), which phosphorylate sphingosine to produce sphingosine 1-phosphate, in kidney fibrosis induced by folic acid (FA) or unilateral ischemia-reperfusion injury. Analysis of Masson trichrome staining and fibrotic marker protein and mRNA expression 14 days after AKI revealed that wild-type (WT) and Sphk1-/- mice exhibited more kidney fibrosis than Sphk2-/- mice. Furthermore, kidneys of FA-treated WT and Sphk1-/- mice had greater immune cell infiltration and expression of fibrotic and inflammatory markers than kidneys of FA-treated Sphk2-/- mice. In contrast, kidneys of Sphk2-/- mice exhibited greater expression of Ifng and IFN-γ-responsive genes (Cxcl9 and Cxcl10) than kidneys of WT or Sphk1-/- mice did at this time point. Splenic T cells from untreated Sphk2-/- mice were hyperproliferative and produced more IFN-γ than did those of WT or Sphk1-/- mice. IFN-γ blocking antibody administered to Sphk2-/- mice or deletion of Ifng (Sphk2-/-Ifng-/- mice) blocked the protective effect of SphK2 deficiency in fibrosis. Moreover, adoptive transfer of Sphk2-/- (but not Sphk2-/-Ifng-/- ) CD4 T cells into WT mice blocked FA-induced fibrosis. Finally, a selective SphK2 inhibitor blocked FA-induced kidney fibrosis in WT mice. These studies demonstrate that SphK2 inhibition may serve as a novel therapeutic approach for attenuating kidney fibrosis.

Endothelial Sphingosine 1­Phosphate Receptor­1 Mediates Protection and Recovery from Acute Kidney Injury.

Epithelial and endothelial injury and a cascade of immune and interstitial cell activation in the kidney lead to AKI. After mild to moderate AKI, the epithelium can regenerate and restore kidney function, yet little is known about the endothelium during these repair processes. Sphingosine 1-phosphate receptor 1 (S1P1), a G protein-coupled receptor, is necessary for vascular homeostasis. Here, we used an inducible genetic approach in a mouse model of AKI, ischemia-reperfusion injury (IRI), to determine the temporal effects of endothelial S1P1 during AKI. Deletion of endothelial S1P1 before IRI exacerbated kidney injury and inflammation, and the delayed deletion of S1P1 after IRI prevented kidney recovery, resulting in chronic inflammation and progressive fibrosis. Specifically, S1P1 directly suppressed endothelial activation of leukocyte adhesion molecule expression and inflammation. Altogether, the data indicate activation of endothelial S1P1 is necessary to protect from IRI and permit recovery from AKI. Endothelial S1P1 may be a therapeutic target for the prevention of early injury as well as prevention of progressive kidney fibrosis after AKI.

Acid sphingomyelinase is activated in sickle cell erythrocytes and contributes to inflammatory microparticle generation in SCD.

Sphingolipids are a class of lipids containing a backbone of sphingoid bases that can be produced de novo through the reaction of palmitate and serine and further metabolized through the activity of various enzymes to produce intermediates with diverse roles in cellular processes and signal transduction. One of these intermediates, sphingosine 1-phosphate (S1P), is stored at high concentrations (1 µM) in red blood cells (RBCs) and directs a wide array of cellular processes mediated by 5 known G-protein coupled receptors (S1P1-S1P5). In this study, we show that RBC membrane alterations in sickle cell disease enhance the activation acid sphingomyelinase by 13%, resulting in increased production and storage of sphingosine (2.6-fold) and S1P (3.5-fold). We also show that acid sphingomyelinase enhances RBC-derived microparticle (MP) generation. These MPs are internalized by myeloid cells and promote proinflammatory cytokine secretion and endothelial cell adhesion, suggesting that potential crosstalk between circulating inflammatory cells and MPs may contribute to the inflammation-rooted pathogenesis of the disease. Treatment with amitriptyline reduces MP generation in vitro and in vivo and might be used to mitigate inflammatory processes in sickle cell disease.

Sphingosine 1-phosphate receptor 3 regulates recruitment of anti-inflammatory monocytes to microvessels during implant arteriogenesis.

Endothelial cells play significant roles in conditioning tissues after injury by the production and secretion of angiocrine factors. At least two distinct subsets of monocytes, CD45(+)CD11b(+)Gr1(+)Ly6C(+) inflammatory and CD45(+)CD11b(+)Gr1(-)Ly6C(-) anti-inflammatory monocytes, respond differentially to these angiocrine factors and promote pathogen/debris clearance and arteriogenesis/tissue regeneration, respectively. We demonstrate here that local sphingosine 1-phosphate receptor 3 (S1P3) agonism recruits anti-inflammatory monocytes to remodeling vessels. Poly(lactic-co-glycolic acid) thin films were used to deliver FTY720, an S1P1/3 agonist, to inflamed and ischemic tissues, which resulted in a reduction in proinflammatory cytokine secretion and an increase in regenerative cytokine secretion. The altered balance of cytokine secretion results in preferential recruitment of anti-inflammatory monocytes from circulation. The chemotaxis of these cells, which express more S1P3 than inflammatory monocytes, toward SDF-1α was also enhanced with FTY720 treatment, but not in S1P3 knockout cells. FTY720 delivery enhanced arteriolar diameter expansion and increased length density of the local vasculature. This work establishes a role for S1P receptor signaling in the local conditioning of tissues by angiocrine factors that preferentially recruit regenerative monocytes that can enhance healing outcomes, tissue regeneration, and biomaterial implant functionality.

Sphingosine 1-phosphate receptor-1 enhances mitochondrial function and reduces cisplatin-induced tubule injury.

Sphingosine 1-phosphate (S1P), the natural sphingolipid ligand for a family of five G protein- coupled receptors (S1P1-S1P5Rs), regulates cell survival and lymphocyte circulation. We have shown that the pan-S1PR agonist, FTY720, attenuates kidney ischemia-reperfusion injury by directly activating S1P1 on proximal tubule (PT) cells, independent of the canonical lymphopenic effects of S1P1 activation on B and T cells. FTY720 also reduces cisplatin-induced AKI. Therefore, in this study, we used conditional PT-S1P1-null (PepckCreS1pr1(fl/fl)) and control (PepckCreS1pr1(w/wt)) mice to determine whether the protective effect of FTY720 in AKI is mediated by PT-S1P1. Cisplatin induced more renal injury in PT-S1P1-null mice than in controls. Although FTY720 produced lymphopenia in both control and PT-S1P1-null mice, it reduced injury only in control mice. Furthermore, the increase in proinflammatory cytokine (CXCL1, MCP-1, TNF-α, and IL-6) expression and infiltration of neutrophils and macrophages induced by cisplatin treatment was attenuated by FTY720 in control mice but not in PT-S1P1-null mice. Similarly, S1P1 deletion rendered cultured PT cells more susceptible to cisplatin-induced injury, whereas S1P1 overexpression protected PT cells from injury and preserved mitochondrial function. We conclude that S1P1 may have an important role in stabilizing mitochondrial function and that FTY720 administration represents a novel strategy in the prevention of cisplatin-induced AKI.

Sphingosine-1-phosphate receptor 1 agonism attenuates lung ischemia-reperfusion injury.

Outcomes for lung transplantation are the worst of any solid organ, and ischemia-reperfusion injury (IRI) limits both short- and long-term outcomes. Presently no therapeutic agents are available to prevent IRI. Sphingosine 1-phosphate (S1P) modulates immune function through binding to a set of G protein-coupled receptors (S1PR1-5). Although S1P has been shown to attenuate lung IRI, the S1P receptors responsible for protection have not been defined. The present study tests the hypothesis that protection from lung IRI is primarily mediated through S1PR1 activation. Mice were treated with either vehicle, FTY720 (a nonselective S1P receptor agonist), or VPC01091 (a selective S1PR1 agonist and S1PR3 antagonist) before left lung IR. Function, vascular permeability, cytokine expression, neutrophil infiltration, and myeloperoxidase levels were measured in lungs. After IR, both FTY720 and VPC01091 significantly improved lung function (reduced pulmonary artery pressure and increased pulmonary compliance) vs. vehicle control. In addition, FTY720 and VPC01091 significantly reduced vascular permeability, expression of proinflammatory cytokines (IL-6, IL-17, IL-12/IL-23 p40, CC chemokine ligand-2, and TNF-α), myeloperoxidase levels, and neutrophil infiltration compared with control. No significant differences were observed between VPC01091 and FTY720 treatment groups. VPC01091 did not significantly affect elevated invariant natural killer T cell infiltration after IR, and administration of an S1PR1 antagonist reversed VPC01091-mediated protection after IR. In conclusion, VPC01091 and FTY720 provide comparable protection from lung injury and dysfunction after IR. These findings suggest that S1P-mediated protection from IRI is mediated by S1PR1 activation, independent of S1PR3, and that selective S1PR1 agonists may provide a novel therapeutic strategy to prevent lung IRI.

Sphingosine Kinase 2 Inhibition and Blood Sphingosine 1-Phosphate Levels.

Sphingosine 1-phosphate (S1P) levels are significantly higher in blood and lymph than in tissues. This S1P concentration difference is necessary for proper lymphocyte egress from secondary lymphoid tissue and to maintain endothelial barrier integrity. Studies with mice lacking either sphingosine kinase (SphK) type 1 and 2 indicate that these enzymes are the sole biosynthetic source of S1P, but they play different roles in setting S1P blood levels. We have developed a set of drug-like SphK inhibitors, with differing selectivity for the two isoforms of this enzyme. Although all SphK inhibitors tested decrease S1P when applied to cultured U937 cells, only those inhibitors with a bias for SphK2 drove a substantial increase in blood S1P in mice and this rise was detectable within minutes of administration of the inhibitor. Blood S1P also increased in response to SphK2 inhibitors in rats. Mass-labeled S1P was cleared more slowly after intravenous injection into SphK2 inhibitor-treated mice or mice lacking a functional SphK2 gene; thus, the increased accumulation of S1P in the blood appears to result from the decreased clearance of S1P from the blood. Therefore, SphK2 appears to have a function independent of generating S1P in cells. Our results suggest that differential SphK inhibition with a drug might afford a method to manipulate blood S1P levels in either direction while lowering tissue S1P levels.

Structure-activity relationship studies of the lipophilic tail region of sphingosine kinase 2 inhibitors.

Sphingosine-1-phosphate (S1P) is a ubiquitous, endogenous small molecule that is synthesized by two isoforms of sphingosine kinase (SphK1 and 2). Intervention of the S1P signaling pathway has attracted significant attention because alteration of S1P levels is linked to several disease states including cancer, fibrosis, and sickle cell disease. While intense investigations have focused on developing SphK1 inhibitors, only a limited number of SphK2-selective agents have been reported. Herein, we report our investigations on the structure-activity relationship studies of the lipophilic tail region of SLR080811, a SphK2-selective inhibitor. Our studies demonstrate that the internal phenyl ring is a key structural feature that is essential in the SLR080811 scaffold. Further, we show the dependence of SphK2 activity and selectivity on alkyl tail length, suggesting a larger lipid binding pocket in SphK2 compared to SphK1.

Dendritic cell sphingosine 1-phosphate receptor-3 regulates Th1-Th2 polarity in kidney ischemia-reperfusion injury.

Dendritic cells (DCs) are central to innate and adaptive immunity of early kidney ischemia-reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1-5), and protection from kidney IRI occurs in S1P3-deficient mice. Through a series of experiments we determined that this protective effect was owing in part to differences between S1P3-sufficient and -deficient DCs. Mice lacking S1P3 on bone marrow cells were protected from IRI, and S1P3-deficient DCs displayed an immature phenotype. Wild-type (WT) but not S1P3-deficient DCs injected into mice depleted of DCs prior to kidney IR reconstituted injury. Adoptive transfer (i.e., i.v. injection) of glycolipid (Ag)-loaded WT but not S1P3-deficient DCs into WT mice exacerbated IRI, suggesting that WT but not S1P3-deficient DCs activated NKT cells. Whereas WT DC transfers activated the Th1/IFN-γ pathway, S1P3-deficient DCs activated the Th2/IL-4 pathway, and an IL-4-blocking Ab reversed protection from IRI, supporting the concept that IL-4 mediates the protective effect of S1P3-deficient DCs. Administration of S1P3-deficient DCs 7 d prior to or 3 h after IRI protected mice from IRI and suggests their potential use in cell-based therapy. We conclude that absence of DC S1P3 prevents DC maturation and promotes a Th2/IL-4 response. These findings highlight the importance of DC S1P3 in modulating NKT cell function and IRI and support development of selective S1P3 antagonists for tolerizing DCs for cell-based therapy or for systemic administration for the prevention and treatment of IRI and autoimmune diseases.

Discovery of Potent, Orally Bioavailable Sphingosine-1-Phosphate Transporter (Spns2) Inhibitors.

Targeting the S1P pathway has resulted in the development of S1P1 receptor modulators for the treatment of multiple sclerosis and ulcerative colitis. We hypothesize that targeting an upstream node of the S1P pathway may provide an improved adverse event profile. In this report, we performed a structure-activity relationship study focusing on the benzoxazole scaffold in SLB1122168, which lead to the discovery of 11i (SLF80821178) as a potent inhibitor of S1P release from HeLa cells (IC50: 51 ± 3 nM). Administration of SLF80821178 to mice induced ∼50% reduction in circulating lymphocyte counts, recapitulating the lymphopenia characteristic of Spns2 null animals. Molecular modeling studies suggest that SLF80821178 binds Spns2 in its occluded inward-facing state and forms hydrogen bonds with Asn112 and Ser211 and π stacking with Phe234. Taken together, SLF80821178 can serve as a scaffold for future inhibitor development and represents a chemical tool to study the therapeutic implication of inhibiting Spns2.

Analogs of FTY720 inhibit TRPM7 but not S1PRs and exert multimodal anti-inflammatory effects.

TRPM7, a TRP channel with ion conductance and kinase activities, has emerged as an attractive drug target for immunomodulation. Reverse genetics and cell biological studies have already established a key role for TRPM7 in the inflammatory activation of macrophages. Advancing TRPM7 as a viable molecular target for immunomodulation requires selective TRPM7 inhibitors with in vivo tolerability and efficacy. Such inhibitors have the potential to interdict inflammatory cascades mediated by systemic and tissue-specialized macrophages. FTY720, an FDA-approved drug for multiple sclerosis inhibits TRPM7. However, FTY720 is a prodrug and its metabolite, FTY720-phosphate, is a potent agonist of sphingosine-1-phosphate (S1P) receptors. In this study, we test non-phosphorylatable FTY720 analogs, which are inert against S1PRs and well tolerated in vivo, for activity against TRPM7 and tissue bioavailability. Using patch clamp electrophysiology, we show that VPC01091.4 and AAL-149 block TRPM7 current at low micromolar concentrations. In culture, they act directly on macrophages to blunt LPS-induced inflammatory cytokine expression, though this likely occurrs through multiple molecular targets. We found that VPC01091.4 has significant and rapid accumulation in the brain and lungs, along with direct anti-inflammatory action on alveolar macrophages and microglia. Finally, using a mouse model of endotoxemia, we show VPC01091.4 to be an efficacious anti-inflammatory agent that arrests systemic inflammation in vivo. Together, these findings identify novel small molecule inhibitors that allow TRPM7 channel inhibition independent of S1P receptor targeting which demonstrate potent, polymodal anti-inflammatory activities ex vivo and in vivo.

Building a better sphingosine kinase-1 inhibitor.

Sphingosine 1-phosphate (S1P) is currently one of the most intensely studied lipid mediators. Interest in S1P has been propelled by the development of fingolimod, an S1P receptor agonist prodrug, which revealed both a theretofore unsuspected role of S1P in lymphocyte trafficking and that such modulation of the immune system achieves therapeutic benefit in multiple sclerosis patients. S1P is synthesized from sphingosine by two SphKs (sphingosine kinases) (SphK1 and SphK2). Manipulation of SphK levels using molecular biology and mouse genetic tools has implicated these enzymes, particularly SphK1, in a variety of pathological processes such as fibrosis, inflammation and cancer progression. The results of such studies have spurred interest in SphK1 as a drug target. In this issue of the Biochemical Journal, Schnute et al. describe a small molecule inhibitor of SphK1 that is both potent and selective. Such chemical tools are essential to learn whether targeting S1P signalling at the level of synthesis is a viable therapeutic strategy.

Sphingosine kinase type 2 inhibition elevates circulating sphingosine 1-phosphate.

S1P (sphingosine 1-phosphate) is a pleiotropic lipid mediator involved in numerous cellular and physiological functions. Of note among these are cell survival and migration, as well as lymphocyte trafficking. S1P, which exerts its effects via five GPCRs (G-protein-coupled receptors) (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases). Although SphK1 is the more intensively studied isotype, SphK2 is unique in it nuclear localization and has been reported to oppose some of the actions ascribed to SphK1. Although several scaffolds of SphK1 inhibitors have been described, there is a scarcity of selective SphK2 inhibitors that are necessary to evaluate the downstream effects of inhibition of this isotype. In the present paper we report a cationic amphiphilic small molecule that is a selective SphK2 inhibitor. In the course of characterizing this compound in wild-type and SphK-null mice, we discovered that administration of the inhibitor to wild-type mice resulted in a rapid increase in blood S1P, which is in contrast with our SphK1 inhibitor that drives circulating S1P levels down. Using a cohort of F2 hybrid mice, we confirmed, compared with wild-type mice, that circulating S1P levels were higher in SphK2-null mice and lower in SphK1-null mice. Thus both SphK1 and SphK2 inhibitors recapitulate the blood S1P levels observed in the corresponding null mice. Moreover, circulating S1P levels mirror SphK2 inhibitor levels, providing a convenient biomarker of target engagement.

International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature.

Lysophospholipids are cell membrane-derived lipids that include both glycerophospholipids such as lysophosphatidic acid (LPA) and sphingoid lipids such as sphingosine 1-phosphate (S1P). These and related molecules can function in vertebrates as extracellular signals by binding and activating G protein-coupled receptors. There are currently five LPA receptors, along with a proposed sixth (LPA1-LPA6), and five S1P receptors (S1P1-S1P5). A remarkably diverse biology and pathophysiology has emerged since the last review, driven by cloned receptors and targeted gene deletion ("knockout") studies in mice, which implicate receptor-mediated lysophospholipid signaling in most organ systems and multiple disease processes. The entry of various lysophospholipid receptor modulatory compounds into humans through clinical trials is ongoing and may lead to new medicines that are based on this signaling system. This review incorporates IUPHAR Nomenclature Committee guidelines in updating the nomenclature for lysophospholipid receptors ( http://www.iuphar-db.org/DATABASE/FamilyMenuForward?familyId=36).

Assay of Sphingosine 1-phosphate Transporter Spinster Homolog 2 (Spns2) Inhibitors.

The sphingosine-1-phosphate (S1P) pathway remains an active area of research for drug discovery because S1P modulators are effective medicine for autoimmune diseases such as multiple sclerosis and ulcerative colitis. As such, other nodes in the pathway can be probed for alternative therapeutic candidates. As S1P elicits its function in an 'outside-in' fashion, targeting the transporter, Spns2, which is upstream of the receptors, is of great interest. To support our medicinal chemistry campaign to inhibit S1P transport, we developed a mammalian cell-based assay. In this protocol, Spns2 inhibition is assessed by treating HeLa, U-937, and THP-1 cells with inhibitors and S1P exported in the extracellular milieu is quantified by LC-MS/MS. Our studies demonstrated that the amount of S1P in the media in inversely proportional to inhibitor concentration. The details of our investigations are described herein.

2-Aminobenzoxazole Derivatives as Potent Inhibitors of the Sphingosine-1-Phosphate Transporter Spinster Homolog 2 (Spns2).

The S1P1 receptor is the target of four marketed drugs for the treatment of multiple sclerosis and ulcerative colitis. Targeting an S1P exporter, specifically Spns2, that is "upstream" of S1P receptor engagement is an alternate strategy that might recapitulate the efficacy of S1P receptor modulators without cardiac toxicity. We recently reported the first Spns2 inhibitor SLF1081851 (16d) that has modest potency with in vivo activity. To develop more potent compounds, we initiated a structure-activity relationship study that identified 2-aminobenzoxazole as a viable scaffold. Our studies revealed SLB1122168 (33p), which is a potent inhibitor (IC50 = 94 ± 6 nM) of Spns2-mediated S1P release. Administration of 33p to mice and rats resulted in a dose-dependent decrease in circulating lymphocytes, a pharmacodynamic indication of Spns2 inhibition. 33p provides a valuable tool compound to explore both the therapeutic potential of targeting Spns2 and the physiologic consequences of selective S1P export inhibition.