Novel target deconvolution in cGAS-STING pathway

cGAS-STING 通路中的新型目标反卷积

• 批准号: 10242612
• 负责人: Jingxin Wang
• 金额: $ 13.9万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242612
• 关键词: Adjuvant; Affinity; Agonist; Alkynes; Applications Grants; Attention; Binding; Binding Proteins; Biology; Biotinylation; CRISPR screen; CRISPR/Cas technology; Cancer Model; Cell Line; Cells; Centers of Research Excellence; Chemicals; Clinical Treatment; Collection; Coupled; Cytosol; Data; Development; Diazomethane; Dimethyl Sulfoxide; Dinucleoside Phosphates; Disease Pathway; Follow-Up Studies; Future; Gel; Genes; Goals; IRF3 gene; Immune response; Immunologic Surveillance; Immunotherapy; In Vitro; Individual; Innate Immune Response; Interferon Type I; Interferons; Knock-out; Knowledge; Label; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Molecular Analysis; Nature; Nucleic Acids; Pathway interactions; Periodicity; Pharmaceutical Preparations; Pharmacology; Phase I Clinical Trials; Phase II Clinical Trials; Phenotype; Publications; Reporter; Reporting; Research Project Grants; Resistance; STING agonists; Signal Transduction; Signaling Molecule; Stimulator of Interferon Genes; T-Cell Activation Pathway; TBK1 gene; Testing; Therapeutic; United States National Institutes of Health; Vaccine Adjuvant; Validation; anti-cancer; base; biophysical analysis; cancer therapy; cell transformation; cellular targeting; crosslink; drug discovery; druggable target; ds-DNA; experimental study; high throughput screening; in vivo Model; insight; interest; knockout gene; member; metabolomics; microbial; new therapeutic target; novel; receptor; response; screening; small molecule

Immunotherapy represents a recent breakthrough in cancer treatment fueled by the accelerating mechanistic understanding of how transformed cells subvert our immunosurveillance. Therefore, developing therapeutic strategies that enhance systemic immunosurveillance in a controllable manner has become one of the major interests in this field. Among different agents, small molecule activators (agonists) of the cGAS-STING pathway have recently attracted attention because they are expected to synergize with immunotherapies and enhance anti-cancer immune response by upregulating the interferon response. We previously identified a compound, BDW568, that was shown to activate the interferon pathway in a STING-dependent manner. Surprisingly, follow-up studies demonstrated that cGAS and STING are not direct target(s) of BDW568. Therefore, observed phenotype suggests that BDW568 acts either through (1) binding to an unknown regulator of the cGAS-STING pathway, or (2) by generating an unknown signaling molecule independent of 2’,3’-cGAMP, the endogenous STING agonist. In the proposed study we will identify the cellular target of BDW568 responsible for the previously discovered phenotype using two orthogonal strategies. In the first approach, we will synthesize BDW568-based photoaffinity probes and use them to label any protein that binds to BDW568. Labeled target(s) will be identified by a whole-cell lysate pulldown experiments coupled with mass spectrometry (MS). In the second approach, we will use CRISPR-Cas9 to individually knock out all the genes that are known to interact with STING or associate with interferon pathways. The knockout of the BDW568 target should demonstrate either resistance to the compound or elevation of the basal interferon level without BDW568 through STING. Any target candidate that emerges from either one of these approaches will be rigorously validated according to the standards of the field to establish that observed phenotype is due to on-target engagement. Successful completion of proposed studies will expand the target space within cGAS-STING pathway. These insights will lead to additional opportunities for developing adjuvant immunotherapies as well as expand our understanding of innate immune response in mammalian cells.

免疫疗法代表了癌症治疗的最新突破， 了解转化细胞如何颠覆我们的免疫监视。因此，开发治疗 以可控制的方式增强全身免疫监视的策略已经成为主要的 在这个领域的兴趣。在不同的药剂中，cGAS-STING的小分子活化剂（激动剂）是优选的。 最近引起了人们的注意，因为它们有望与免疫疗法协同作用， 通过上调干扰素反应增强抗癌免疫反应。 我们先前鉴定了一种化合物BDW 568，该化合物被证明可以激活干扰素通路。 依赖的方式。令人惊讶的是，后续研究表明，cGAS和STING并不直接 BDW 568的目标。因此，观察到的表型表明BDW 568通过以下方式起作用：（1）结合 cGAS-STING途径的未知调节物，或（2）通过产生未知信号分子 独立于内源性STING激动剂2 '，3'-cGAMP。在拟议的研究中，我们将确定 细胞靶BDW 568负责先前发现的表型使用两个正交 战略布局在第一种方法中，我们将合成基于BDW 568的光亲和探针，并使用它们来 标记与BDW 568结合的任何蛋白质。将通过全细胞裂解物下拉法鉴别标记的靶标 实验结合质谱法（MS）。在第二种方法中，我们将使用CRISPR-Cas9来 单独敲除已知与STING相互作用或与干扰素相关的所有基因 途径。BDW 568靶标的敲除应证明对该化合物的抗性，或 通过STING，在没有BDW 568的情况下，基础干扰素水平升高。任何出现的目标候选人 将根据该领域的标准严格验证这些方法中的任何一种， 确定所观察到表型是由于中靶接合（on-target engagement）。圆满完成拟议的 研究将扩大cGAS-STING途径内的靶空间。这些见解将导致额外的 开发辅助免疫疗法的机会，以及扩大我们对先天性免疫缺陷的理解。 哺乳动物细胞的免疫反应。