Dual Covalent Targeting of STING Cysteines 292/309 Disrupts Functional Oligomerization and Enables Potent Antagonist Development

ABSTRACT Dysregulated STING activation is a well‐established driver of pathological inflammation in autoimmune and autoinflammatory diseases, underscoring the need for targeted therapeutic inhibition. Current STING antagonist development has predominantly relied on phenotypic screening strategies. In contrast, we introduce a rational design strategy that directly disrupts STING signaling at its structural origin by covalently targeting cysteine residues within the C‐terminal domain (CTD) to prevent functional oligomerization. Through covalent warhead repurposing, we identified P005091, previously known as a USP7 inhibitor, as a STING antagonist that operates via a non‐classical nucleophilic displacement mechanism. Mechanistic investigation demonstrated that inhibition by P005091 depends on its concurrent engagement of Cys292 and Cys309, as evidenced by the fact that its activity to block STING oligomerization was abolished only by the C292A/C309A double mutation. Functionally, P005091 potently suppressed STING signaling and type I interferon responses in vitro and in vivo. Structure‐guided optimization yielded the advanced compounds NTP14 and NTP16, which exhibit markedly enhanced cellular potency and robust efficacy in ameliorating type I interferon‐driven pathology in multiple preclinical models, including DSS‐induced colitis. Our work establishes dual covalent CTD targeting as a transformative strategy for STING antagonist development and opens a new therapeutic avenue for quenching STING‐driven inflammation at its source.