Novel target deconvolution in cGAS-STING pathway

免疫疗法代表了由加速机制推动的癌症治疗的最新突破 了解转化的细胞如何颠覆我们的免疫监控。因此，发展治疗性 以可控的方式加强系统免疫监督的策略已成为主要的 对这一领域的兴趣。在不同的药物中，cGAS-STING的小分子激活剂(激动剂) 通路最近引起了人们的注意，因为它们有望与免疫疗法和 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，它被证明能激活干扰素途径。 叮咬成瘾的方式。令人惊讶的是，后续研究表明，cGAS和刺痛不是直接的 BDW568的目标(S)。因此，观察到的表型表明BDW568通过(1)结合发挥作用 通过产生未知的信号分子，或者(2)通过产生未知的信号分子 不依赖内源性刺激剂2‘，3’-cGAMP。在拟议的研究中，我们将确定 用两个正交法研究BDW568细胞靶点与先前发现的表型 战略。在第一种方法中，我们将合成基于BDW568的光亲和探针，并将它们用于 标记任何与BDW568结合的蛋白质。标记的目标(S)将通过全细胞裂解物下拉来识别 实验结合质谱仪(MS)。在第二种方法中，我们将使用CRISPR-Cas9来 单独敲除所有已知与刺痛或与干扰素相关的基因 小路。BDW568靶基因的敲除应该表现出对该化合物的抗性或 无BDW568的基础干扰素水平通过STING升高。任何出现的目标候选人 任何一种方法都将根据现场标准进行严格验证，以 确定观察到的表型是由于目标参与。成功完成建议的 研究将拓展cGAS-STING途径的靶向空间。这些见解将导致额外的 发展佐剂免疫疗法的机会以及扩大我们对先天免疫的理解 哺乳动物细胞的免疫反应。

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.