Novel target deconvolution in cGAS-STING pathway

cGAS-STING 通路中的新型目标反卷积

• 批准号: 10434460
• 负责人: Jingxin Wang
• 金额: $ 21.68万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-06 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434460
• 关键词: Adjuvant; Agonist; Attention; Binding; Binding Proteins; CRISPR/Cas technology; Cells; Coupled; Disease Pathway; Follow-Up Studies; Genes; Immune response; Immunologic Surveillance; Immunotherapy; Individual; Innate Immune Response; Interferons; Knock-out; Label; Mammalian Cell; Mass Spectrum Analysis; Molecular Analysis; Pathway interactions; Phenotype; Resistance; STING agonists; Signaling Molecule; Stimulator of Interferon Genes; Therapeutic; anti-cancer; base; cancer therapy; cell transformation; cellular targeting; experimental study; insight; interest; novel; response; 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，这是对转化细胞如何颠覆我们的免疫监视方式的加速机理理解。因此，开发治疗 以受控方式增强系统性免疫监视的策略已成为该领域的主要利益之一。在不同的药物中，CGAS刺激途径的小分子激活剂（激动剂）最近引起了人们的注意，因为它们有望通过上调干扰素反应来与免疫疗法协同并增强抗癌免疫响应。 我们先前鉴定了一种化合物BDW568，该化合物以s依赖性方式激活了干扰素途径。令人惊讶的是，后续研究表明，CGA和Sting不是BDW568的直接靶标。因此，观察到的表型表明，BDW568通过（1）与CGAS丁字途径的未知调节剂结合，或（2）通过产生独立于2'，3'-cgamp，内源性刺痛激动剂而产生未知的信号分子。在拟议的研究中，我们将使用两种正交策略确定负责先前发现的表型的BDW568的细胞靶标。在第一种方法中，我们将合成基于BDW568的光亲和力问题，并使用它们标记任何与BDW568结合的蛋白质。标记的靶标将通过与质谱法（MS）结合的全细胞裂解物下拉螺丝实验来识别。在第二种方法中，我们将使用CRISPR-CAS9单独淘汰所有已知与STING相互作用或与干扰素途径相关的基因。 BDW568目标的敲除应证明对基本干扰素水平的抗性或升高，而没有BDW568通过STING。从任何一种方法中出现的任何目标候选者都将根据该领域的标准进行严格验证，以确定观察到的表型是由于目标参与。成功完成拟议的研究将扩大CGAS刺入途径内的目标空间。这些见解将为开发可调节的免疫疗法以及我们对哺乳动物细胞中先天免疫的理解提供更多机会。