Cellular Target Deconvolution of Small Molecules Using a Selection-Based Genetic Screening Platform

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.