Small molecule in vivo probe development targeting the IL-6/STAT3 pathway for potential multiple sclerosis therapy

 描述(申请人提供)：多发性硬化症(MS)是美国年轻人创伤后神经功能障碍的主要原因，因此大多数患者在成年后的大部分时间都受到多发性硬化症的影响。目前的多发性硬化症治疗是部分有效的，因此有必要开发创新的策略。白介素6(IL-6)通过转录因子STAT3传递信号，在MS发病的多个途径中发挥核心作用，并且在MS患者中发现了IL-6/STAT3信号的异常。首先，在MS的EAE模型中，IL-6通过STAT3信号诱导高脑源性IL-17的产生，产生Th17细胞，转移严重的疾病；同时，IL-6抑制可诱导T调节细胞(ITreg)的产生，iTreg是抑制致病性炎症T细胞反应的关键。结果，TJeff/Treg平衡向过多的T效应反应倾斜，有利于自身免疫的发展。此外，IL-6/STAT3信号通路参与了MS患者TJeff细胞对Treg介导的抑制的抵抗，从而进一步破坏了TJeff/Treg的平衡。综上所述，这些研究表明，IL-6/STAT3信号通路可能成为逆转MS患者发病机制的创新靶点。此外，口服小分子化合物通常比常用的基于多肽/蛋白质的药物具有更好的生物利用度和制造特性。此外，当使用口服药物时，治疗依从性得到改善。为此，我们打算开发针对IL-6/STAT3途径的小分子候选药物。我们开发了四个新的小分子化合物，MDL-5和MDL-16靶向IL-6；LLL-12和LY-5靶向STAT3。MDL-5/16与gp130的D1域结合，阻止IL-6/IL-6R/gp130信号复合体形成的六角化过程中IL-6/gp130的相互作用；Lll-12/LY-5结合STAT3的SH2结构域，阻止STAT3的磷酸化和二聚化。当添加到细胞培养中时，所有四种先导化合物都显著抑制IL-6诱导的髓鞘特异性CD4T细胞中IL-17的产生。基于前人的研究和我们的初步数据，我们假设新的小分子IL-6/STAT3抑制剂可以修复CD4T反应的TJeff/Treg失衡，并抑制MS EAE模型的疾病发展和进展。以下目的将解决这一假说。目的1.优化作为候选药物的化合物。目的2.在MS EAE模型中，检测新型小分子IL-6/STAT3抑制剂对修复髓鞘特异性CD4T细胞的TJeff/Treg失衡和抑制疾病发展的作用。本研究是首次尝试使用新的小分子来调节IL-6/STAT3信号，并将在MS的EAE模型中阐明IL-6信号调节小鼠和人的CD4T反应和体内疾病发展的机制。该研究将为未来使用针对IL-6/STAT3信号的新型药理化合物的临床研究奠定基础，最终目标是治疗多发性硬化。

 DESCRIPTION (provided by applicant): Multiple Sclerosis (MS) is the leading cause of neurologic disability in the United States in young adults after trauma, thus most patients suffer from the effects of MS for most of their adult life. The current MS treatments are partially effective, making it necessary to develop innovative strategies. Interleukin-6 (IL-6), signaling through transcription factor STAT3, shares a central role in multiple pathways of MS pathogenesis and dysregulated IL-6/STAT3 signaling has been shown in MS patients. First, IL-6, signaling through STAT3, induces the generation of highly encephalitogenic IL-17, producing Th17 cells that transfers severe disease in the EAE model of MS. Meanwhile, IL-6 suppresses the generation of inducible T regulatory cells (iTreg), which is critical for dampening pathogenic inflammatory T cell responses. As a result, the Teff/Treg balance is skewed towards excessive T effector responses, favoring the development of autoimmunity. Furthermore, IL-6/STAT3 signaling contributes to the resistance of Teff cells to Treg-mediated suppression in MS patients, which further impairs Teff/Treg balance. Altogether, these studies suggest that the IL-6/STAT3 signaling pathway may serve as an innovative target for reversing pathogenesis in MS patients. In addition, orally available small molecule compounds usually offer improved bioavailability and manufacturing features over commonly used peptide/protein-based drugs. Moreover, therapy adherence is improved when oral agents are used. To this end, we intend to develop small molecule drug candidates targeting IL- 6/STAT3 pathway. We have developed four novel small molecule compounds, MDL-5 and MDL-16 targeting IL-6; LLL-12 and LY-5 targeting STAT3. MDL-5/16 bind to the D1 domain of GP130, preventing the IL-6/GP130 interaction during the hexamerization step of IL-6/IL- 6R/GP130 signaling complex formation; LLL-12/LY-5 bind to the SH2 domain of STAT3, preventing STAT3 phosphorylation and dimerization. When added into cell culture, all four lead compounds significantly inhibit IL-6 induced IL-17 production in myelin-specific CD4 T cells. Based on these previous studies and our preliminary data, we hypothesize that novel small molecule IL-6/STAT3 inhibitors repair the Teff/Treg imbalance of CD4 T responses and suppress disease development and progression in the EAE model of MS. The following aims will address this hypothesis. Aim 1. Optimize the compounds as drug candidates. Aim 2. Determine the effects of novel small molecule IL-6/STAT3 inhibitors on repairing the Teff/Treg imbalance of myelin-specific CD4 T responses and on suppressing disease development in the EAE model of MS. Aim 3. Determine the effects of novel small molecular IL-6/STAT3 inhibitors on repairing Teff/Treg balance in CD4 T cells from MS patients. This study is the first attempt to modulate IL-6/STAT3 signaling using novel small molecules and will elucidate the mechanisms through which IL-6 signaling regulates murine and human CD4 T responses and disease development in vivo in the EAE model of MS. This study will establish the basis for future clinical studies using novel pharmacological compounds that target IL-6/STAT3 signaling, with the ultimate goal of treatment of multiple sclerosis.