Development of antagonists targeting STING in systemic lupus erythematosus

项目摘要 干扰素基因刺激物(STING)是一种胞浆内质网锚定受体蛋白 在先天免疫的繁殖中，通过产生干扰素来感应胞浆DNA(干扰素)。 机制研究表明，在肿瘤微环境中产生干扰素可导致激活 肿瘤抗原特异性CD8+T细胞免疫可导致肿瘤消退。刺法激活刺法 激动剂应在肿瘤微环境中产生先天T细胞介导的抗肿瘤免疫，并具有 作为癌症治疗药物的巨大潜力。相反，我们假设：抑制刺痛将导致 干扰素的产生减少，这将降低对胞浆DNA和RNA的免疫反应，从而 减少系统性红斑狼疮(SLE)危及生命的情况。分子动力学(MD) 人类HAQ、REF和野生型(WT)刺等位基因的平衡晶体结构被聚类以找到 利用刚性进行计算对接筛选计算化学文库的最佳构象 受体、诱导配对和量子极化配基模型。刺痛激动剂和拮抗剂的模型 都被开发出来了。一种不以环二核苷酸为基础的新型低分子有机分子 (如STING的正常配体2‘，3’-环-GAMP)被发现是STING的强结合体。该化合物 在实验室合成，其结构经LC/MS、1H、13C核磁共振确证。蛋白质 代表HAQ和WT等位基因(占人类人口的78.3%)的测试 我们的化合物分别以2‘，3’-cGAMP和DMSO为阳性对照和阴性对照 记者模特儿。简而言之，pIRF-3(由STING激活的直接下游蛋白)是通过 THP-1单核细胞的发光，并给出了荧光素发光的信号，即 比2‘，3’-cGAMP弱约100倍。此外，在表面等离子体激元共振(SPR) 实验确定我们的化合物具有~400 nm的Kd。我们假设我们的 化合物是一种部分激动剂，可以使用迭代计算轮次将其转化为完全拮抗剂 建模、综合和实验测试。 我们正在设计这种化合物的类似物，作为治疗SLE的潜在的刺痛拮抗剂。强者 在探索可以抑制刺痛的R基团修饰的同时，化合物的结合将被保留 系统性红斑狼疮的途径。值得注意的是，刺痛的拮抗剂预计将在改善 系统性红斑狼疮有危及生命的情况。需要的是验证性实验，我们的先导化合物可以 被优化为刺痛的拮抗剂，将在SLE中有效。我们正在利用我们的专业知识在 计算机药物发现，合成和药物化学，生物物理结合测量，以及 以体外细胞为基础的分析，以执行“先导探索”研究，将导致化合物可以在 在参与全面的铅优化倡议之前，狼疮易感小鼠需要确定其疗效。

Project Summary Stimulator of interferon genes (STING) is a cytosolic endoplasmic reticulum anchored receptor protein involved in the propagation of innate immune sensing of cytosolic DNA through the production of Interferon-ß (IFN-ß). Mechanistic studies have shown IFN-ß production within a tumor microenvironment can result in activation of tumor antigen-specific CD8+ T-cell immunity that can lead to tumor regression. STING activation by STING agonists should result in innate T-cell mediated anti-tumor immunity in the tumor microenvironment and have significant potential as a cancer therapeutic. Conversely, we hypothesize that: inhibition of STING will lead to a decreased production of IFN-ß which will reduce the immune response to cytosolic DNA and RNA and thereby reduce life-threatening conditions in systemic lupus erythematosus (SLE). Molecular Dynamics (MD) equilibrated crystal structures for human HAQ, REF, and wild type (WT) STING alleles were clustered to find optimal conformations for computational chemical library screening via computational docking utilizing rigid receptor, induced fit, and quantum polarized ligand models. Models for both STING agonists and antagonists were developed. A novel low-molecular-weight organic molecule that is not based on a cyclic dinucleotide (such as STING’s normal ligand, 2’,3’-cyclic-GAMP) was found as a strong binder of STING. The compound was synthesized in our laboratory and its structure was confirmed using LC/MS and 1H and 13C NMR. Proteins representing both the HAQ and WT alleles (representing 78.3% of the human population) were tested against our compound with 2’,3’-cGAMP and DMSO as positive and negative controls, respectively in a luciferase reporter model. In short, pIRF-3 (the immediate downstream protein activated by STING) was measured by luminescence in THP-1 monocytic leukemic cells and gave a signal for the luminescence of luciferin that was approximately 100 fold weaker than 2’,3’-cGAMP. Moreover, in Surface Plasmon Resonance (SPR) experiments we determined that our compound possesses a KD of ~400nM. We hypothesize that our compound is a partial agonist that can be converted to a full antagonist using iterative rounds of computational modeling, synthesis, and experimental testing. We are designing analogs of this compound as potential antagonists of STING for SLE therapy. The strong binding of the compound will be preserved while exploring R group modifications that can suppress the STING pathway for SLE. It is noteworthy that antagonists of STING are anticipated to play a strong role in ameliorating life threatening conditions in SLE. What is needed are confirmatory experiments that our lead compound can be optimized as a STING antagonist that will be effective in SLE. We are employing our expertise in computational drug discovery, synthetic and medicinal chemistry, biophysical binding measurements, and in vitro cell-based assays, to perform “lead exploration” studies that will result in compounds that can be tested in lupus-prone mice to determine their efficacy before engaging in a full blown lead optimization initiative.