Novel target deconvolution in cGAS-STING pathway

cGAS-STING 通路中的新型目标反卷积

基本信息

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
项目类别：
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

项目摘要

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，加速的机制推动了这一突破了解转化细胞如何破坏我们的免疫监视，从而开发治疗方法。以可控方式增强系统免疫监视的策略已成为主要的策略之一在这个领域的兴趣中，cGAS-STING 的小分子激活剂（激动剂）。途径最近引起了人们的关注，因为它们有望与免疫疗法产生协同作用，并且通过上调干扰素反应来增强抗癌免疫反应。我们之前发现了一种化合物 BDW568，它被证明可以激活细胞中的干扰素通路。令人惊讶的是，后续研究表明 cGAS 和 STING 不是直接的。因此，观察到的表型表明 BDW568 通过 (1) 结合发挥作用。 cGAS-STING 通路的未知调节因子，或 (2) 通过生成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 BDW568 的细胞靶标负责先前使用两个正交发现的表型在第一种方法中，我们将合成基于 BDW568 的光亲和探针并使用它们来标记任何与 BDW568 结合的蛋白质将通过全细胞裂解物下拉来识别。在第二种方法中，我们将使用 CRISPR-Cas9 进行实验与质谱 (MS) 相结合。单独敲除所有已知与 STING 相互作用或与干扰素相关的基因 BDW568 靶标的敲除应表现出对该化合物的抗性或没有 BDW568 通过 STING 提高基础干扰素水平任何出现的候选目标。这些方法中的任何一种都将根据该领域的标准进行严格验证确定观察到的表型是由于成功完成了拟议的目标。这些研究将扩大 cGAS-STING 通路中的目标空间。开发辅助免疫疗法以及扩大我们对先天性的理解的机会哺乳动物细胞的免疫反应。