High-Throughput Screening and Initial SAR Studies Identify a Novel Sub-micromolar Potent Human cGAS Inhibitor

Abstract

Cyclic GMP-AMP synthase (cGAS) has emerged as a promising therapeutic target of several human diseases, including Alzheimers disease (AD) and other neurodegenerative disorders. As a cytosolic DNA sensor, cGAS generates an innate immune response to promote neuroinflammation by producing an endogenous agonist of the stimulator of interferon genes (STING), 23-cyclic GMP-AMP (cGAMP), which activates the cGAS-STING pathway. We have performed a high-throughput screening of a chemical library containing over 300K small molecules at the Fisher Drug Discovery Resource Center (DDRC), Rockefeller University (RU), to identify multiple hit inhibitors of human (h)-cGAS. We used a modified Kinase Glo(R) Luminescent Kinase assay, which was earlier developed at RU and later used by multiple groups, including ours, to perform primary screening of the library using h-cGAS. The hit candidates bearing novel scaffolds are structurally diverse and exhibited in vitro activity in the low micromolar range. RU-0610270 or compound (cpd) 1, a sulfonamide derivative, is one of the most potent hits (IC50=1.88 {micro}M), selected for hit expansion and structure-activity relationship (SAR) analysis. We synthesized new analogs of cpd 1 and evaluated them in vitro against h-cGAS to identify cpd 6 (IC50=0.66 {micro}M) as the most potent hit analog. We further profiled cpd 6 and found that it modestly inhibited cGAMP levels by 29% at 30 {micro}M in THP1 cells without detectable toxicity, and by 76% at 100 {micro}M, albeit with a moderate decrease ([~]20%) in cell viability. These results highlight a novel chemical series with promising in vitro activity, providing a starting point for the development of selective and potent human cGAS inhibitors for clinical use.