Controlling the flux of sphingosine-1-phosphate in vivo

1-磷酸鞘氨醇(S1P)趋化梯度对于免疫细胞的正确时空定位是必需的，而血液-组织S1P梯度支持内皮屏障的完整性。我们现在建议提供一种通过改变淋巴-淋巴S1P梯度来调节免疫系统的新方法，S1P梯度对淋巴细胞运输至关重要。S1P梯度在免疫细胞迁移中起关键作用的最初迹象是发现了免疫抑制药物Fingolimod的作用机制。这种药物使淋巴细胞S1P1受体减敏，使这些细胞无法从次级淋巴组织迁移到传出淋巴的S1P丰富环境。尽管Fingolimod和其他S1P1受体激动剂最终作为一种药物取得了成功，但它们具有靶向的心脏和血管毒性，这就需要额外的策略来通过操纵S1P信号来调节免疫反应。对突变小鼠的研究预测，一个可行的替代策略是通过抑制从内皮细胞向传出淋巴供应S1P的转运蛋白Spns2来消除淋巴-淋巴S1P梯度。然而，目前还没有测试这一想法所需的Spns2抑制剂。作为满足这一需求的第一步，我们利用酿酒酵母中高水平S1P的毒性建立了S1P转运蛋白的检测方法。我们使用这种方法来筛选我们的S1P激动剂和鞘氨醇激酶抑制剂的重点化学库，并确定了一种命中化合物，在最小的化学操作之后，它导致了一种先导化合物，该化合物可以驱动淋巴细胞减少和血浆S1P的降低，预期Spns2抑制剂是这样的。通过迭代的化学合成和药理测试，我们将优化我们的主要Spns2抑制剂，并发现和优化其他化学系列的Spns2抑制剂。最终，我们将生产出具有适合体内应用的药代动力学特性的强效Spns2抑制剂。Spns2抑制剂的选择性将通过对其他S1P相互作用蛋白的严格反筛选来确定，这些蛋白包括S1P受体、分解代谢和合成代谢酶以及另一种红细胞特异性的S1P转运蛋白Mfsd2b。在开发Spns2抑制剂时，我们将使用血浆S1P水平和外周血淋巴细胞计数作为靶向结合的生物标志物。Spns2抑制剂将被部署在一系列表明免疫调节的疾病模型中。此外，我们将评估我们的Spns2抑制剂与S1P信号相关的潜在不良事件，包括血管泄漏、心动过缓和耳毒性。最理想的是，我们的研究将验证Spns2作为免疫系统调节的治疗靶点。至少，我们将为探索S1P的复杂生物学提供可靠的化学工具。

Sphingosine 1-phosphate (S1P) chemotactic gradients are necessary for correct temporal and spatial positioning of immune cells while the blood-tissue S1P gradient supports endothelial barrier integrity. We propose now to provide a new method of modulating the immune system by changing the lymph-lymph node S1P gradient, which is crucial for lymphocyte trafficking. The initial indication that S1P gradients have a pivotal role in immune cell migration was the discovery of the mechanism of action of the immunosuppressive drug, fingolimod. This drug desensitizes lymphocyte S1P1 receptors, which renders these cells unable to migrate from secondary lymphoid tissues to the S1P rich environment of efferent lymph. Although ultimately successful as a medicine, fingolimod and other S1P1 receptor agonists have on-target cardiac and vascular toxicities, which necessitates additional strategies to modulate the immune response by manipulating S1P signaling. Studies with mutant mice predict that a viable alternative strategy is to eliminate the lymph-lymph node S1P gradient by inhibiting the transporter, Spns2, which supplies S1P from endothelial cells to efferent lymph. However, the Spns2 inhibitors that are required to test this idea are not available. As a first step in meeting this need, we took advantage of the toxicity of high levels of S1P in Saccharomyces cerevisiae to build an S1P transporter assay. We used this assay to screen our focused chemical library of S1P agonists and sphingosine kinase inhibitors and identified a hit compound that, after minimal chemical manipulation, resulted in a lead compound that drives the lymphopenia and reduction in plasma S1P expected of an Spns2 inhibitor. Through iterative chemical synthesis and pharmacologic testing, we will optimize our lead Spns2 inhibitor as well as discover and optimize additional chemical series of Spns2 inhibitors. Ultimately, we will generate potent Spns2 inhibitors with the pharmacokinetic properties suitable for in vivo applications. The selectivity of the Spns2 inhibitors will be ascertained by rigorous counter-screening against other S1P interacting proteins including S1P receptors, catabolic and anabolic enzymes and another, erythrocyte-specific, S1P transporter, Mfsd2b. In developing Spns2 inhibitors, we will use plasma S1P levels and peripheral blood lymphocyte counts as biomarkers of target engagement. Spns2 inhibitors will be deployed in a battery of disease models where immune-modulation is indicated. Further, we will assess our Spns2 inhibitors for potential adverse events associated with S1P signaling including vascular leak, bradycardia and ototoxicity. Optimally, our studies will validate Spns2 as a therapeutic target for immune system modulation. At a minimum, we will provide reliable chemical tool for exploring the complex biology of S1P.