Validating Inhibitors of the Immunomodulatory Sphingosine 1-Phosphate Transporter SPNS2

项目摘要 信号脂质鞘氨醇1-磷酸（S1 P）在免疫系统中起着关键作用。值得注意的是， S1 P调节淋巴细胞从淋巴结中的排出，淋巴细胞最初在淋巴结中被激活以对抗病原体， 进入淋巴，淋巴将细胞运送到血液中，使它们能够到达受感染的组织。FTY 720，一种药物 靶向包括S1 PR 1在内的四种S1 P受体，成为FDA批准的第一种口服治疗多种 硬化症通过阻止致病性T细胞离开淋巴器官，FTY 720阻止它们进入 中枢神经系统靶向S1 PR 1的第二代药物在II期也显示出了希望 牛皮癣和结肠炎的临床试验不幸的是，这些药物也靶向内皮细胞中的S1 PR 1， 心肌细胞，导致严重的副作用，包括黄斑水肿和心动过缓。我们发现 主要易化超家族转运蛋白SPNS 2将S1 P供应到淋巴而不是血液中，使其成为一种有前途的 新的药物靶点，这将使空间特异性调制S1 P信号。此外，我们的数据 表明Spns 2是活化T细胞在炎症部位积累和缺失所必需的， SPNS 2在实验性自身免疫性脑脊髓炎中具有保护作用， 硬化症基于这些观察结果，我们着手开发一种高通量筛选，以识别小 Spns 2活性的分子调节剂。我们开发并优化了一种快速发射高通量质量 使用基于光谱的测定来检测由Spns 2表达细胞分泌到培养基中的S1 P。利用该 在一项分析中，我们筛选了Evotec的252，454种化合物发现文库，产生了5，899种抑制剂，截止值为11.5% （5σ）。随后，我们挑选了1962个点击进行确认筛选，得到923个确认点击。这里我们 建议开发检测方法并验证HTS工作中出现的命中。与纽约大学办公室 我们已经确定了一个顾问和合同研究组织团队， 在检测开发和筛选、命中验证以及合成和药物化学方面的专长， 与我们的实验室合作，开发和执行命中验证方案，产生高质量的探针靶向 Spns2。这些工具化合物可用于探测Spns 2的生物学，并有可能首先在 类药物，用于治疗自身免疫性疾病。

Project Summary The signaling lipid sphingosine 1-phosphate (S1P) plays critical roles in the immune system. Notably, S1P regulates lymphocyte exit from lymph nodes, where lymphocytes are initially activated to fight pathogens, out into lymph, which carries the cells to blood and enables them to travel to infected tissues. FTY720, a drug that targets four S1P receptors including S1PR1, became the first FDA-approved oral therapeutic for multiple sclerosis. By blocking pathogenic T cells from exiting lymphoid organs, FTY720 prevents them from accessing the central nervous system. Second-generation drugs that target S1PR1 have also shown promise in Phase II clinical trials for psoriasis and colitis. Unfortunately, these drugs also target S1PR1 in endothelial cells and cardiomyocytes, resulting in serious side effects including macular edema and bradycardia. We found that the major facilitator superfamily transporter SPNS2 supplies S1P into lymph but not blood, making it a promising new drug target that would enable spatially specific modulation of S1P signaling. Furthermore, our data indicate that Spns2 is required for the accumulation of activated T cells at sites of inflammation and deleting Spns2 has a protective effect in experimental autoimmune encephalomyelits, a mouse model of multiple sclerosis. Based on these observations, we set out to develop a high throughput screen to identify small molecule modulators of Spns2 activity. We developed and optimized a Rapid-Fire high-throughput mass spectrometry-based assay to detect S1P secreted into the media by Spns2-expressing cells. Utilizing this assay, we screened Evotec's 252,454 compound Discovery Library, yielding 5,899 inhibitors at a cut-off of 11.5% (5σ). We subsequently cherry picked 1962 hits for confirmation screens, yielding 923 confirmed hits. Here, we propose to develop assays and validate the hits that emerged from the HTS effort. Together with NYU's Office of Therapeutics Alliances we have identified a team of consultants and contract research organizations with expertise in assay development and screening, hit validation, and synthetic and medicinal chemistry that will work with our laboratory to develop and execute a hit validation scheme yielding high quality probes targeting Spns2. These tool compounds can be used to probe the biology of Spns2 and have the potential to yield first in class drugs for treating autoimmune diseases.