Combining chemoproteomics and phenotypic screening for identifying STING modulators

The cGAS–STING pathway is a crucial component of the innate immune system that functions to detect the presence of cytosolic DNA and, in response, trigger the expression of inflammatory genes, therefore playing a critical role in autoimmune diseases, inflammation, and cancer. Yet, there are no approved STING inhibitors in the clinic. By high-throughput phenotypic screening of thousands of small covalent molecule fragments, we were able to identify >30 compounds that inhibit STING pathway activation. To understand the compound"s mechanism we investigated the cysteines to which the compounds bind using activity-based protein profiling (ABPP), followed by an in-depth quantitative whole proteome and phospho-proteome analyses to identify the proteome-wide changes by the compounds. Although different compounds demonstrated varying levels of STING modulation, by the profiling of the cysteines to which the compounds bind, we identified that many of them target STING cysteine 91 (C91), resulting in inhibition of STING palmitoylation. The phospho-proteome and whole proteome analyses complemented this observation and validated the changes resulting from STING inhibition.

In addition to identifying the binding site of the compounds, the cysteine profiling approach revealed changes in the reactivity of other cysteines. We observed changes in the reactivity of STING C148 and C206, suggesting higher availability of these positions to the desthiobiotin iodoacetamide probe due to STING conformational changes. As well as changes in the availability of cysteines in STING interactors, for example, IRF3 C371 which resides in the proximity of STING-IRF3 interaction. These examples demonstrate the utility of this approach for the understanding of the global changes in protein interactions and conformation by the compounds.

Identifying structurally variable compounds in the screen directs us to improve the specificity of the compounds to STING. In addition, ABPP results enable us to identify off target proteins to which the compounds bind. Together, this information led us to design a library of elaborated compounds and tested their binding specificity by ABPP and their activity using the functional assay, and identifying compounds with improved specificity and binding to STING.

The combination of high-throughput phenotypic screening and proteomic approaches facilitates the identification of functionally relevant inhibitors and their mechanisms.