Novel Macrolide Th17 Inhibitors

描述（由申请人提供）：Th 17细胞及其细胞因子产物IL-17 A和IL-17 F与慢性炎性疾病（包括囊性纤维化、COPD和类固醇抗性哮喘）有关。此外，这些细胞因子与自身免疫性疾病如多发性硬化症和类风湿性关节炎有关。抑制Th 17细胞因子产生的抗IL-12 p40抗体（优特克单抗）最近已被批准用于治疗银屑病;然而，这种药物也可以抑制Th 1免疫，理论上增加细胞内病原体感染的风险。为了鉴定可能对Th 17细胞而不是Th 1细胞具有选择性的小分子，我们最近进行了化合物文库筛选以鉴定将抑制Th 17细胞而不是Th 1细胞的小分子。在该筛选中，我们包括已知的化合物，如钙调磷酸酶抑制剂（环孢菌素A）、FK-506和雷帕霉素，它们是已知的T细胞功能抑制剂。CyA、FK- 506和雷帕霉素抑制Th 1和Th 17细胞，因此是非选择性的。然而，大环内酯类药物，特别是阿奇霉素显示剂量依赖性抑制Th 17的发展，ED 50约为100 5 M，几乎没有抑制Th 1的反应。在此基础上，我们还研究了克拉霉素和罗红霉素的活性。后者化合物也显示出显著的抑制Th 17发育的活性，ED 50为200 5 M。基于这些数据，我们假设，这些大环内酯类代表潜在的抑制剂的Th 17效应器功能和结构活性关系（SAR）可以用来提高这些化合物的活性。我们将以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，以鉴定活性所需的结构和更有效的Th 17细胞因子加工抑制剂。以红霉素为起始原料合成了一系列大环内酯类化合物。然后将这些化合物与不同剂量的Th 17或Th 1细胞一起孵育，然后用抗CD 3/抗CD 28活化。结果指标包括细胞内IL-17和IFN？以及通过FACS测定细胞活力。将收获细胞上清液，并检测分泌的IL-17或IFN？将由Luminex测量。具体目标2。使用Th 17/Th 1肺部炎症模型体内筛选目标1中鉴定的有效化合物。如前所述，将使用卵清蛋白特异性Th 17或Th 1细胞IV在过继转移模型中体内测试具有强效IC 50的选定化合物。结果的措施将包括BAL嗜中性粒细胞和巨噬细胞迁移以及肺水平的IL-17，IFN？以及下游Th 17和Th 1靶基因（分别为KC、G-CSF、MCP-1和Th 2、IP-10）。这一系列的实验将大大增加我们对Th 17细胞小分子抑制剂潜力的认识。这些化合物可以改善慢性肺部疾病如COPD或类固醇抵抗性哮喘的治疗。 公共卫生相关性：白细胞介素-17可导致肺部炎症和哮喘。该项目旨在鉴定能够抑制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.