De novo design of protein condensation inhibitors by targeting an allosteric site of cGAS.

Cyclic GMP-AMP synthase (cGAS), a key mediator of the cGAS-STING DNA sensing pathway that triggers type-I interferon responses, plays a crucial role in innate immunity and has been implicated in the pathogenesis of various disease. Despite advances in the development of cGAS inhibitors, none have reached the market and there remains an unmet need for divergent chemical scaffolds with high selectivity, potency across species, and target-adaptive mechanisms of action to explore cGAS's potential as a therapeutic target. Here we report the structural, biochemical, cellular, and mechanistic characterization of the XL series of allosteric inhibitors, designed to engage an innovative allosteric site near the activation loop of cGAS. Among them, XL-3156 and XL-3158 emerge as potent, selective, cross-species cGAS inhibitors that simultaneously occupy allosteric and orthosteric sites, stabilizing the activation loop in a closed, inactive conformation and thereby attenuating the cGAS-DNA interactions. Moreover, these allosteric inhibitors, also known as protein condensation inhibitors (PCIs), significantly suppress cGAS-DNA condensate formation, triggering a morphological transition from liquid-solid phase separation (LSPS) to liquid-liquid phase separation (LLPS) at the molecular level while eliminating LLPS in cells. The distinct mechanism of action enables PCIs to achieve synergistic effects in combination with orthosteric inhibitors. These results establish a mechanism-driven pharmacological strategy to inhibit cGAS through PCIs that modulate phase separation primarily by engagement of the allosteric site.