Novel Macrolide Th17 Inhibitors

描述（由申请人提供）：Th17细胞及其细胞因子产物IL-17A和IL-17F与慢性炎症性疾病有关，包括囊性纤维化、COPD和类固醇抵抗性哮喘。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Th17细胞因子细化的抗il -12p40抗体（Ustekinumab）最近已被批准用于治疗牛皮癣；然而，这种药物也可以抑制Th1免疫，理论上会增加细胞内病原体感染的风险。为了鉴定可能对Th17细胞而不是Th1细胞有选择性的小分子，我们最近进行了一个化合物文库筛选，以鉴定可以抑制Th17细胞而不抑制Th1细胞的小分子。在这个筛选中，我们包括了已知的化合物，如钙调磷酸酶抑制剂（环孢素A）， FK-506和雷帕霉素，它们是已知的t细胞功能抑制剂。CyA、FK- 506和雷帕霉素均抑制Th1和Th17细胞，因此具有非选择性。然而，大环内酯类药物，特别是阿奇霉素，对Th17的抑制表现出剂量依赖性，ED50约为100 5M，对Th1的抑制作用很小。在此基础上，测定了克拉霉素和罗红霉素的活性。后一种化合物也显示出显著的抑制Th17发育的活性，ED50为200 5M。基于这些数据，我们假设这些大环内酯类化合物代表Th17效应功能的潜在抑制剂，并且结构活性关系（SAR）可以用来提高这些化合物的活性。我们将用两个具体目标来检验这一假设。具体目标大环内酯类似物的合成和SAR分析，以确定Th17细胞因子活性所需的结构和更有效的抑制剂。以红霉素为原料，合成一系列大环内酯类似物。然后将这些化合物以不同剂量与Th17或Th1细胞孵育，然后用抗cd3 /抗cd28激活。结果测量将包括细胞内IL-17和IFN？流式细胞仪检测细胞活力。收集细胞上清液，分泌IL-17或IFN的水平？将由Luminex测量。具体目标2。使用Th17/Th1肺炎症模型在体内筛选Aim 1中鉴定的有效化合物。如前所述，选择具有有效ic50的化合物将在卵清蛋白特异性Th17或Th1细胞IV的过继转移模型中进行体内测试。结果测量将包括BAL中性粒细胞和巨噬细胞迁移以及肺IL-17、IFN？以及下游的Th17和Th1靶基因（分别为KC、G-CSF、MCP-1和MIG、IP-10）。提出的一系列实验应该大大增加我们对Th17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慢性阻塞性肺病或类固醇抵抗性哮喘的治疗。

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.