Validating Inhibitors of the Immunomodulatory Sphingosine 1-Phosphate Transporter SPNS2

项目摘要 信号转导脂鞘氨醇1-磷酸(S1P)在免疫系统中起着关键作用。值得注意的是， S1P调节淋巴细胞从淋巴结出，在那里淋巴细胞最初被激活以对抗病原体， 进入淋巴，将细胞运送到血液中，使它们能够到达受感染的组织。FTY720，一种药物 该药物针对包括S1PR1在内的四种S1P受体，成为FDA批准的第一种针对多发性硬化的口服疗法 硬化症。FTY720通过阻止致病T细胞离开淋巴器官，阻止它们访问 中枢神经系统。针对S1PR1的第二代药物在第二阶段也显示出希望 牛皮癣和结肠炎的临床试验。不幸的是，这些药物也针对内皮细胞中的S1PR1和 心肌细胞，导致严重副作用，包括黄斑水肿和心动过缓。我们发现， 主要促进剂超家族转运体SPNS2将S1P供应到淋巴中，但不是血液中，使其成为一种有希望的 新的药物靶点将使S1P信号的空间特异性调制成为可能。此外，我们的数据 表明Spns2是激活的T细胞在炎症和缺失部位聚集所必需的 Spns2对实验性自身免疫性脑脊髓炎的保护作用 硬化症。基于这些观察，我们着手开发一种高通量屏幕来识别小分子 Spns2活性的分子调节剂。我们开发并优化了一种快速点火高通量质量 用分光光度法检测表达Spns2的细胞分泌的S1P。利用这一点 实验中，我们筛选了Evotec的252,454个化合物发现文库，以11.5%的截止值产生了5899个抑制剂 (5σ)。随后，我们精心挑选了1962个点击量用于确认屏幕，产生了923个确认点击量。在这里，我们 建议开发化验并验证HTS工作中出现的命中结果。与纽约大学办公室合作 在治疗联盟中，我们已经确定了一个顾问团队和合同研究组织， 在分析开发和筛选、HIT验证以及合成和药物化学方面的专业知识 与我们的实验室合作开发和执行HIT验证方案，以产生高质量的探针 Spns2.这些工具化合物可用于探测Spns2的生物学，并有可能在 治疗自身免疫性疾病的一类药物。

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.