Novel Macrolide Th17 Inhibitors

描述(申请人提供)：Th17细胞及其细胞因子产物IL-17A和IL-17F与囊性纤维化、慢性阻塞性肺疾病和激素抵抗型哮喘等慢性炎症性疾病有关。此外，这些细胞因子还与自身免疫性疾病有关，如多发性硬化症和类风湿性关节炎。最近，抑制Th17细胞因子分泌的抗IL-12p40抗体(Ustekinumab)已被批准用于银屑病的治疗；然而，这种药物也可以抑制Th1免疫，理论上增加了感染细胞内病原体的风险。为了确定可能对Th17细胞而不是Th1细胞具有选择性的小分子，我们最近进行了一次化合物文库筛选，以确定可以抑制Th17细胞而不是Th1细胞的小分子。在这项筛选中，我们纳入了已知的化合物，如钙调神经磷酸酶抑制剂(环孢素A)、FK-506和雷帕霉素，它们是已知的T细胞功能抑制物。CyA、FK-506和雷帕霉素同时抑制Th1和Th17细胞，因此是非选择性的。然而，大环内酯类化合物，特别是阿奇霉素，显示出剂量依赖性地抑制Th17的发展，ED50约为100 5M，而对Th1反应的抑制很小。在此基础上，我们还检测了克拉霉素和罗红霉素的活性。后一类化合物还具有明显的抑制Th17发育的活性，其ED50为200 5M。基于这些数据，我们假设这些大环内酯类化合物是Th17效应功能的潜在抑制剂，结构活性关系(SAR)可以用来提高这些化合物的活性。我们将通过两个具体目标来检验这一假设。具体目标1.大环内酯类似物的合成和SAR分析，以确定活性所需的结构和更有效的Th17细胞因子形成抑制物。以红霉素为原料合成了一系列大环内酯类似物。然后，这些化合物将以不同的剂量与Th17或Th1细胞孵育，然后用抗CD3/抗CD28激活。结果指标将包括细胞内IL-17和干扰素？用流式细胞仪检测细胞存活率。将收获的细胞上清和分泌的IL-17或干扰素水平？将由Luminex测量。特殊目的2.使用Th17/Th1肺部炎症模型在体内筛选目标1中确定的有效化合物。如前所述，选定的具有有效IC50的化合物将在体内使用卵白蛋白特异性Th17或Th1细胞IV进行体内测试。观察指标包括BAL中性粒细胞数、巨噬细胞移行率以及肺组织IL-17、干扰素？以及下游的Th17和Th1靶基因(分别为KC、G-CSF、MCP-1和MIG、IP-10)。这一系列拟议的实验应该会极大地增加我们对Th17细胞小分子抑制物潜力的了解。这些化合物可能会改善慢性肺部疾病的治疗，如COPD或类固醇抵抗型哮喘。 与公共健康相关：白介素17可引起肺部炎症和哮喘。这个项目是为了识别可以抑制IL-17和肺部炎症的药物。

DESCRIPTION (provided by applicant): Th17 cells and their cytokine products, IL-17A and IL-17F have been implicated in chronic inflammatory diseases including cystic fibrosis, COPD and steroid resistant asthma. Furthermore these cytokines have been implicated in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Anti-IL-12p40 antibodies (Ustekinumab) which suppress Th17 cytokine elaboration have recently been approved for the treatment of psoriasis; however this drug can also suppress Th1 immunity and theoretically increase the risk of infection with intracellular pathogens. To identify small molecules which may be selective for Th17 cells as opposed to Th1 cells, we recently conducted a compound library screen to indentify small molecules that would suppress Th17 cells but not Th1 cells. In this screen we included known compounds such as calcineurin inhibitors (Cyclosporine A), FK-506, and rapamycin, which are known suppressors of T-cell function. CyA, FK- 506, and rapamycin suppressed both Th1 and Th17 cells and thus were non-selective. However macrolides, specifically Azithromycin showed dose dependent suppression of Th17 development with an ED50 of approximately 100 5M and little suppression of Th1 responses. Based on this we also examined the activities of clarithromycin and roxithromycin. The latter compounds also showed significant activity in inhibiting Th17 development with an ED50 of 200 5M. Based on these data, we hypothesize that these macrolides represent potential inhibitors of Th17 effector function and that structure activity relationships (SAR) can be used to improve the activity of these compounds. We will test this hypothesis with two specific aims. Specific Aim 1. Synthesis and SAR analysis of macrolide analogs to identify structures required for activity and more potent inhibitors of Th17 cytokine elaboration. A series of macrolide analogs will be synthesized using erythromycin as the starting material. These compounds will then be incubated with Th17 or Th1 cells at varying doses prior to activation with anti-CD3/anti-CD28. Outcome measures will include intracellular IL-17 and IFN? as well as cell viability by FACS. Cell supernatants will be harvested and the level of secreted IL-17 or IFN? will be measured by Luminex. Specific Aim 2. In vivo screening of potent compounds identified in Aim 1 using a Th17/Th1 model of lung inflammation. Selected compounds with potent IC50s will be tested in vivo in an adoptive transfer model using ovalbumin specific Th17 or Th1 cells IV as previously described. Outcome measures will include BAL neutrophilia and macrophage emigration as well as lung levels of IL-17, IFN? as well as the downstream Th17 and Th1 target genes (KC, G-CSF, MCP-1 and MIG, IP-10 respectively). The series of proposed experiments should greatly increase our knowledge of the potential of small molecule inhibitors of Th17 cells. These compounds may improve treatment for chronic lung diseases such as COPD or steroid resistant asthma. PUBLIC HEALTH RELEVANCE: Interleukin-17 can cause lung inflammation and asthma. This project is to indentify drugs that can inhibit IL-17 and lung inflammation.