Controlling sphingosine 1-phosphate synthesis and trafficking

鞘氨醇激酶(SphK1，SphK2)催化形成一种重要的细胞外介质， 1-磷酸鞘氨醇(S1P)。S1P生物学的一个基本方面是S1P的巨大差异 血液或淋巴(高)和组织(低)之间的丰度，称为S1P血管梯度。这 梯度维持血管内皮细胞屏障功能，促进淋巴样淋巴细胞动员 纸巾。事实上，S1P1受体激动剂药物(如Fingolimod)在治疗上是有益的，因为S1P 信号转导对S1P梯度的变化高度敏感。我们使用我们的SphK2抑制剂来证明 在合成水平阻断S1P信号通过减缓S1P使S1P血管梯度变陡 从血迹中清除。这一结果表明，SphK2抑制剂将在治疗 内皮屏障受损的情况，例如急性肾损伤和脓毒症。虽然我们的 最近发现的SphK2抑制剂在体内是活性的，在效力、口服利用度和化学物质方面都有改善 需要多样性才能将它们推向临床。我们将通过产生更多的 在我们目前的化学支架上添加了缓蚀剂，并开发了一种新的第二种支架。当前的脚手架 也产生了一些SphK1抑制剂，但这些药物在小鼠SphK1上缺乏效力，这排除了它们的测试 在一些关键疾病模型上的有效性。与SphK2相反，抑制Sphk1减少了S1P血管 为了探索由此产生的生理后果，需要多种抑制剂。我们将使用 反复几轮合成和测试以生成SphK1抑制剂文库，重点是增加 它们在小鼠SphK1上的效力和发现具有适当药代动力学特性的抑制剂 啮齿动物。了解SphK抑制的分子机制，并为合成 化学策略，我们将用X射线解决这两种同工酶与结合抑制剂的结构 结晶学。最后，我们将发现S1P导出器的拦截器SPNS2，它将S1P提供给 淋巴，从而维持淋巴细胞从淋巴中流出所需的S1P血管梯度 从器官到淋巴。目前，由于SPNS2抑制剂不可用，这种特殊的方法可以 对S1P梯度的操纵和随后的免疫调节仍然完全没有被探索。这个 我们课程的优势在于化学(Santos)和药理学(Lynch)的结合。 现在我们再加上结构生物学(法厄姆)。我们的中心主题是理解治疗 在合成(SphK抑制)或运输水平上操纵S1P梯度的可能性 (SPNS2封锁)。我们的工作效率记录使我们能够从根本上洞察S1P 生物学，例如，我们发现SphK2抑制调节S1P信号以保护内皮功能，这是一种新的 治疗策略。现在，我们提出了极大改进的发展和详细表征 SphK抑制剂，并制作必要的化学工具，以审问SPNS2作为潜在的药物靶点。

Sphingosine kinases (SphK1, SphK2) catalyze the formation of an important extracellular mediator, sphingosine 1-phosphate (S1P). A fundamental aspect of S1P biology is the large difference in S1P abundance between blood or lymph (high) and tissue (low), which is termed the S1P vascular gradient. This gradient maintains vascular endothelial barrier function and facilitates lymphocyte mobilization from lymphoid tissues. Indeed, S1P1 receptor agonist drugs (e.g. fingolimod) are therapeutically beneficial because S1P signaling is highly sensitive to changes in S1P gradient. We used our SphK2 inhibitors to demonstrate that interdicting S1P signaling at the level of synthesis steepens the S1P vascular gradient by slowing S1P clearance from the blood. This result suggests that SphK2 inhibitors will be extremely useful in treating conditions where the endothelial barrier is compromised, e.g. acute kidney injury and sepsis. Although our recently discovered SphK2 inhibitors are active in vivo, improvements in potency, oral availability and chemical diversity are needed to advance them to the clinic. We will accomplish these goals by generating additional inhibitors on our current chemical scaffold and by developing a novel second scaffold. The current scaffold has also yielded a few SphK1 inhibitors but these lack potency at mouse SphK1, which precludes their testing for efficacy in some key disease models. In contrast to SphK2, inhibition of Sphk1 decreases the S1P vascular gradient and to probe the resulting physiological consequences, multiple inhibitors are needed. We will use iterative rounds of synthesis and testing to generate a library of SphK1 inhibitors with emphases on increasing their potency at mouse SphK1 and discovering inhibitors that have suitable pharmacokinetic properties in rodents. To understand the molecular mechanism of SphK inhibition as well as to inform the synthetic chemistry strategies, we will solve the structures of both isozymes with bound inhibitors using X-ray crystallography. Finally, we will discover a blocker of the S1P exporter, SPNS2, which provides the S1P to lymph and thereby maintains the S1P vascular gradient that is required for lymphocyte egress from lymphoid organs to lymph. Currently, due to the unavailability of SPNS2 inhibitors, this particular approach to the manipulation of S1P gradient and subsequent immunomodulation remains completely unexplored. The strength of our program is the synergism in the combination of chemistry (Santos) and pharmacology (Lynch) to which we now add structural biology (Faham). Our central theme of is to understand the therapeutic potential of manipulating the S1P gradients either at the level of synthesis (SphK inhibition) or transport (SPNS2 blockade). We have a track record of productivity that enabled a fundamental insight into S1P biology, e.g. our discovery that SphK2 inhibition modulates S1P signaling to protect endothelial function, a new therapeutic strategy. Now, we propose the development and detailed characterization of greatly improved SphK inhibitors and to make the chemical tools necessary to interrogate SPNS2 as a potential drug target.