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

Abstract

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.

Structural Evaluations of a Selective Human STINGA230 Agonist and Its Use in Macrophage Immunotherapies

“…BDW568 is required to be hydrolyzed by CES1 to yield the carboxylic acid metabolite to interact with STING A230 . 25 We tested other esters, such as ethyl (15) and isopropyl (16) esters, 25 and observed a slightly weakened activity (Figure 5).…”

“…We previously demonstrated that STING activation in THP-1 cells using BDW568 is solely dependent on A230, but not H71 or Q293. 25 The crystal structure of STING A230 -CTD-BDW-OH showed that there is a small unoccupied cavity on the dimethyl thiophene side in BDW568 (Figure 3A). To probe the size of the cavity, we linked the 4,5dimethyl groups on the thiophene ring (A) with one carbon (1) or two carbons (2) (Figure 4).…”

“…No activity of BDW568 against STING G230 (major allele) was observed, suggesting a nearly complete selectivity in naturally occurring STING variants. 25 BDW568 is a methyl ester and can be quickly hydrolyzed by cytosolic carboxylesterase 1 (CES1) after cellular uptake to yield the active metabolite BDW-OH (Figure 1). 25 Here, we report the crystal structure of the STING A230 C-terminal domain (CTD)-BDW-OH complex and structure−activity relationship (SAR) studies.…”

“…25 BDW568 is a methyl ester and can be quickly hydrolyzed by cytosolic carboxylesterase 1 (CES1) after cellular uptake to yield the active metabolite BDW-OH (Figure 1). 25 Here, we report the crystal structure of the STING A230 C-terminal domain (CTD)-BDW-OH complex and structure−activity relationship (SAR) studies. We also validate the activity of BDW568 in primary human cells from healthy individuals with G230 or A230 alleles or purified primary macrophages with a transduced exogenous STING A230 allele.…”

Innovative CRISPR Screening Promotes Drug Target Identification

“…Such pooled CRISPR screening largely relies on positive selection after offsetting the antiproliferative effects of the drugs and thus cannot be applied to nonantiproliferative small molecules. In a recent issue of ACS Central Science , Jingxin Wang and co-workers 3 established a loss-of-function CRISPR screening platform for target identification of a nonantiproliferative drug candidate by linking the compound activation pathway with the expression of a suicide gene. Using this platform, they pinpointed STING as the target of BDW568, a small molecule IFN-I activator, and identified a key metabolizing enzyme, CES1, that activates BDW568 in cells.…”