Zhao Junxing, Tang Zhichao, Selvaraju Manikandan, Johnson Kristen A, Douglas Justin T, Gao Philip F, Petrassi H Michael, Wang Michael Zhuo, Wang Jingxin
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66047, United States.
Calibr, Scripps Research Institute, La Jolla, California 92037, United States.
ACS Cent Sci. 2022 Oct 26;8(10):1424-1434. doi: 10.1021/acscentsci.2c00609. Epub 2022 Sep 22.
Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways.
小分子药物靶点鉴定是表型药物发现中必不可少且常常是限速的步骤，仍然是一项重大挑战。在此，我们报告了一种新型平台，通过利用成簇规律间隔短回文重复序列（CRISPR）敲除文库的力量来鉴定信号通路激活剂的靶点。该平台将自杀基因的表达与小分子激活的信号通路相联系，以创建一个筛选系统。利用这个系统，使用CRISPR单导向（sg）RNA文库进行功能缺失筛选能够正向富集靶点被敲除的细胞。然后通过测序揭示感兴趣的小分子活性所需的药物靶点和其他必需基因的身份。我们在新发现的I型干扰素信号激活剂BDW568上测试了这个平台，确定干扰素基因刺激因子（STING）为其靶点，羧酸酯酶1（CES1）是激活BDW568以实现靶点结合所需的关键代谢酶。我们在此展示的平台可以成为一种通用方法，适用于鉴定激活不同信号通路的多种小分子的靶点。
