Small molecule inhibition of human cGAS reduces total cGAMP output and cytokine expression in cells

Caroline Wiser,Byungil Kim,Manuel Ascano,Jessica Vincent

doi:10.1038/s41598-020-64348-y

Abstract

The cGAS-STING pathway is a major mechanism that mammalian cells utilize to detect cytoplasmic dsDNA from incoming viruses, bacteria, or self. CYCLIC GMP-AMP SYNTHASE (cGAS) is the sensor protein that directly binds dsDNAs. cGAS synthesizes cyclic GMP-AMP (cGAMP), which binds to the adaptor STIMULATOR OF INTERFERON GENES (STING), activating an INTERFERON REGULATORY FACTOR 3 (IRF3)-mediated immune response. Constitutive activation can result in interferonopathies such as Aicardi-Goutieres Syndrome (AGS) or other lupus-like autoimmune disorders. While inhibitors targeting mouse or human cGAS have been reported, the identification of a small molecule that targets both homologs of cGAS has been challenging. Here, we show that RU.521 is capable of potently and selectively inhibiting mouse and human cGAS in cell lines and human primary cells. This inhibitory activity requires the presence of cGAS, but it cannot suppress an immune response in cells activated by RNA, Toll-like receptor ligands, cGAMP, or recombinant interferon. Importantly, when RU.521 is applied to cells, the production of dsDNA-induced intracellular cGAMP is suppressed in a dose-dependent manner. Our work validates the use of RU.521 for probing DNA-induced innate immune responses and underscores its potential as an ideal scaffold towards pre-clinical development, given its potency against human and mouse cGAS.

Highlights

The deployment of pattern recognition receptors (PRRs) is the primary method for the innate immune system to detect pathogens or cellular damage via their associated molecular patterns (PAMPs and DAMPs)[1]
THP-1 cells are a monocyte-like human cell line that expresses CYCLIC GMP-AMP SYNTHASE (cGAS) and STIMULATOR OF INTERFERON GENES (STING), which are required for sensing cytoplasmic dsDNA and eliciting INTERFERON REGULATORY FACTOR 3 (IRF3)-mediated INTERFERON BETA 1 (IFNB1) upregulation
We found that RU.[521] could inhibit endogenous Human CYCLIC GMP-AMP SYNTHASE (h-cGAS) in WT THP-1 cells that were stimulated with herring testis DNA (HT-DNA) (Fig. 1a right panel and Fig. 1b)[11,50]

Summary

Introduction

The deployment of pattern recognition receptors (PRRs) is the primary method for the innate immune system to detect pathogens or cellular damage via their associated molecular patterns (PAMPs and DAMPs)[1]. Knockout studies have shown that Trex[1] null mice, which have elevated interferon levels, return to WT levels of interferon upon knockout of cGAS38,39, STING40,41, or IRF340 These studies further validate the importance of cGAS and its role in regulating immunity, and it has become a prime candidate for the development of small molecules that activate or inhibit its immunopotent activity[42,43,44,45]. We demonstrate that RU.[521] inhibits cytoplasmic DNA-dependent upregulation of IRF3-dependent transcriptional targets only in the presence of an intact cGAS-STING pathway. We exposed THP-1 cells to various PAMPs while in the presence of RU.[521], and demonstrate that there are no observable off-target effects – indicating that RU.[521] only suppresses innate immune activation induced by dsDNA if cGAS is present. Our data illustrate the utility of RU.[521] in inhibiting both mouse and human cGAS and its potential as a molecular scaffold to be further developed for eventual clinical use

Methods

Results

Conclusion