Homo sapiens Raw sequence reads

Small-molecule drug target deconvolution is essential in phenotypic drug discovery and remains a major challenge in the field. Here, we report BDW568, a novel type-I interferon signalling activator identified from high-throughput screening, and a cost-effective, label-free platform that was used to identify BDW568's cellular target. This target identification platform links the expression of a suicide gene to the BDW568-activated pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches the cells with the drug target knockout. The identities of BDW568's target, stimulator of interferon genes (STING), and other essential genes to the drug action are then uncovered by sequencing. We also discovered carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for STING protein binding. The platform we present here can be adapted to a wide range of other drugs that activate gene expression.