An iguratimod prodrug reduces RA-FLS invasiveness by disrupting STAT1–C3–TNFα-mediated crosstalk between fibroblast-like synoviocytes and macrophages

Background: Rheumatoid arthritis (RA) is driven in part by hyperactivated fibroblast-like synoviocytes (FLS) that invade articular structures. Iguratimod (IGU), a conventional synthetic DMARD, is clinically effective, but its direct molecular target and impact on synovial cell-cell crosstalk remain unclear. We aimed to elucidate how IGU regulates FLS invasiveness and inflammatory signaling, identify its upstream target within the JAK-STAT pathway, and develop a prodrug with improved pharmacokinetics while preserving disease-modifying activity. Methods: We combined in vitro assays in MH7A cells and rat RA-FLS with RNA sequencing and conditioned-medium fast-astral DIA proteomics to characterize IGU's effects on TNF-α-induced migration, invasion, and signaling. STAT1 dependence was interrogated by siRNA knockdown, phosphorylation-deficient mutant reconstitution and IFN-γ rescue. Integrated single-cell RNA-seq of RA and healthy synovium, together with CellChat analysis and complement component 3a (C3a) stimulation of THP-1-derived macrophages, was used to define FLS-macrophage crosstalk. Reverse virtual screening, molecular docking, thermal shift assays, cellular thermal shift assays, kinase assays, and molecular dynamics simulations were applied to characterize IGU-TYK2 interactions. A sulfonamide N-acyl IGU prodrug (AD811) was rationally designed and evaluated for pH-dependent stability, plasma and microsomal metabolism, pharmacokinetics, efficacy, and short-term safety in collagen-induced arthritis rats. Results: IGU suppressed TNF-α-induced FLS migration and invasion without cytotoxicity by selectively inhibiting STAT1 Y701 phosphorylation and nuclear translocation, while sparing STAT1 Y727 phosphorylation and STAT2 Y690 phosphorylation. Bulk and single-cell transcriptomic analyses revealed STAT1 hyperactivation in RA lining-layer FLS and uncovered a STAT1-C3-TNFα feedback loop in which FLS-derived C3/C3a enhances macrophage TNF-α production, thereby reinforcing FLS activation; IGU disrupted this loop by reducing STAT1 activity, C3 transcription, and C3a-driven macrophage TNF-α induction. Biochemical and biophysical studies showed that IGU directly engages the TYK2 JH2 pseudokinase domain, alters its thermal behavior, and inhibits kinase activity of a TYK2 construct containing JH2 and JH1, while not measurably inhibiting the isolated JH1 catalytic domain, consistent with JH2-dependent allosteric modulation of TYK2 output. The prodrug AD811 exhibited pH-sensitive stability, rapid plasma conversion to IGU, favorable oral bioavailability, and therapeutic efficacy, joint protection, and preliminary hepatic and gastric safety comparable to equimolar IGU in vivo. Conclusion: In FLS-centered experimental systems, IGU reduces RA-FLS invasiveness by targeting TYK2 JH2 and disrupting a STAT1-C3-TNFα feedback loop between lining-layer FLS and macrophages, thereby attenuating both intrinsic fibroblast aggressiveness and inflammatory crosstalk. The prodrug AD811 maintains these disease-modifying actions while improving pharmacokinetic properties, nominating AD811 as a promising candidate for further translational development in RA. The translational potential of this article: This study mechanistically links IGU, a clinically used csDMARD, to selective modulation of TYK2 JH2 and downstream STAT1 signaling in synovial lining-layer FLS. By showing that, in FLS-centered models, IGU disrupts a STAT1-C3-TNFα feedback loop between FLS and macrophages and thereby reduces FLS invasiveness and inflammatory crosstalk, our data provide a concrete cellular and molecular basis for its disease-modifying effects in rheumatoid arthritis. Furthermore, the rationally designed prodrug AD811 exhibits improved pharmacokinetic properties and a favorable short-term safety profile in vivo, supporting its further evaluation as a potential oral small-molecule candidate.