Development of antagonists targeting STING in systemic lupus erythematosus

系统性红斑狼疮靶向 STING 拮抗剂的开发

基本信息

批准号：
10078933
负责人：
Wayne Guida
金额：
$ 15.56万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10078933
关键词：
Agonist Alleles Animals Antinuclear Antibodies Autoantibodies Autoimmune Diseases Binding Bioavailable Biological Assay Biological Availability Biophysics CD8-Positive T-Lymphocytes Cell Extracts Cell physiology Cells Complex Computer Analysis Computer Models Core Facility Crystallization DNA DNA Binding Development Dimethyl Sulfoxide Dinucleoside Phosphates Docking Dose Endoplasmic Reticulum Future Gene Activation Glomerulonephritis Health Heart Heart failure Human IRF3 gene Immune response Immune system Immunity Immunology In Vitro Interferon-beta Kidney Kidney Failure Laboratories Lead Leukemic Cell Life Ligands Luciferases Lupus Malignant Neoplasms Measurement Measures Mediating Modeling Modification Molecular Conformation Molecular Weight Monitor Monocytic leukemia Mus Pathway interactions Patients Periodicity Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Play Population Production Proteins Proteinuria Quality of life RNA Reporter Research Design Role Scanning Services Signal Transduction Stimulator of Interferon Genes Structure Surface Plasmon Resonance Synthesis Chemistry Systemic Lupus Erythematosus T-Lymphocyte Testing Therapeutic Tissues Toxicology Tumor Antigens Tumor Immunity Western Blotting X-Ray Crystallography analog anti-dsDNA antibodies base chemical synthesis design drug discovery efficacy study experimental study flexibility improved in vivo innate immune sensing lead optimization liquid chromatography mass spectrometry luciferin luminescence lupus prone mice molecular dynamics mouse model novel pharmacokinetics and pharmacodynamics preservation promoter quantum receptor scaffold screening small molecule libraries tumor tumor microenvironment virtual web site

项目摘要

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.

项目摘要干扰素基因（STING）的刺激剂是涉及的胞质内质网的受体蛋白通过产生干扰素 - ß（IFN-ß），胞质DNA的先天免疫传感在传播中。机械研究表明，肿瘤微环境内的IFN-ß产生可能导致激活肿瘤抗原特异性CD8+ T细胞免疫，可导致肿瘤退化。通过刺激激活激活激动剂应导致肿瘤微环境中的先天T细胞介导的抗肿瘤免疫作为癌症疗法的巨大潜力。相反，我们假设：抑制sting将导致 IFN-ß产生的降低，这将减少对胞质DNA和RNA的免疫响应，从而减少减少系统性红斑狼疮（SLE）的威胁生命的条件。分子动力学（MD）人类HAQ，REF和野生型（WT）sting等位基因的平衡晶体结构被聚集以找到使用刚性通过计算对接进行计算化学库筛选的最佳构象接收器，诱导的拟合和量子偏光配体模型。刺痛者和拮抗剂的模型被开发了。一种新型的低分子量有机分子，不是基于环状二核苷酸的（例如Sting的正常配体，2'，3'cycle-gamp）被认为是刺痛的强壮粘合剂。化合物在我们的实验室中合成，并使用LC/MS以及1H和13C NMR确认其结构。蛋白质代表HAQ和WT等位基因（占人口的78.3％）的测试我们的化合物分别在荧光素酶中分别为阳性对照和阴性对照记者模型。简而言之，通过 THP-1单核细胞性白血病细胞中的发光，并给出了Luciferin的发光信号大约比2'，3'-cgamp弱100倍。此外，在表面等离子体共振（SPR）中实验我们确定我们的化合物具有〜400nm的KD。我们假设我们的化合物是一种部分激动剂，可以使用迭代回合的计算转换为完整的拮抗剂建模，合成和实验测试。我们将这种化合物的类似物设计为SLE治疗的潜在拮抗剂。坚强探索可以抑制刺痛的R组修改时，将保留化合物的结合 SLE的途径。值得注意的是，刺的对手预计将在改善方面发挥重要作用 SLE中威胁生命的条件。需要的是确认的实验，我们的铅化合物可以被优化为在SLE中有效的Sting拮抗剂。我们正在利用我们的专业知识计算药物发现，合成和医学化学，生物物理结合测量以及基于体外细胞的测定，进行“铅探索”研究，该研究将导致可以在浓厚的小鼠在进行完整吹铅优化计划之前确定其效率。