Identification of a regulatory allosteric site in Arid5a unveils a therapeutic axis for systemic inflammation.

Adenine-thymine (AT)-rich interactive domain-containing protein 5a (Arid5a) is an RNA-binding protein (RBP) that post-transcriptionally stabilizes mRNAs encoding proinflammatory mediators, including Interleukin-6 (IL-6), thereby amplifying inflammation. However, structural basis and regulatory mechanisms of Arid5a function remain poorly defined. In this study, we identified and characterized a conserved allosteric site within the ARID domain that regulates Arid5a-mediated mRNA stabilization. Using integrative in silico modeling and in vitro assays, 2 picolinamide-based inhibitors, NFP1 and NFP2, were designed to specifically engage the allosteric site constituent residues. Mutational and functional analyses revealed Tyr88 and Val91 as key regulatory elements within the allosteric site, essential for transmitting conformational regulation upon ligand engagement. Pharmacological inhibition of the allosteric site disrupted Arid5a interaction with target RNA stem-loop structure, reduced stability of Il6 mRNA, and attenuated inflammatory responses in Lipopolysaccharide (LPS)-stimulated macrophages. Furthermore, Arid5a inhibitors reduced the stability of other target mRNAs, including Signal transducer and activator of transcription 3 (Stat3) and OX40, in polarized T helper 17 (Th17) cells. In a murine model of LPS-induced septic shock, treatment with NFP1 or NFP2 significantly improved survival, reduced clinical severity, and mitigated tissue damage in vital organs. These findings identify a new mechanism regulating Arid5a activity and present Arid5a allosteric inhibition as a promising therapeutic strategy for managing systemic inflammation including sepsis.