• First identification of prostaglandin E synthase 3 (Ptges3) as the direct covalent target of andrographolide (AP). • Discovery of a novel allosteric binding site (Cys58) that enables dual inhibition of Ptges3′s enzymatic and chaperone functions. • AP binding disrupts the Ptges3-Hsp90-NF-κB axis, suppressing PGE2 production and mitigating pulmonary fibrosis. • Genetic validation via Ptges3 knockdown confirms target specificity and necessity for AP’s antifibrotic efficacy Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by chronic inflammation and fibroblast activation, with limited treatment options. The bioactive diterpenoid andrographolide (AP) exhibits anti-inflammatory and antifibrotic properties, but its direct molecular targets and precise mechanism remain unclear. This study aimed to identify the primary functional target of AP and elucidate the molecular mechanism underlying its therapeutic effects against pulmonary fibrosis. We employed activity-based protein profiling (ABPP) to identify covalent targets of AP in macrophages. Functional validation was performed using siRNA knockdown, enzymatic assays, molecular docking, and biophysical analyses. The therapeutic efficacy and target dependency of AP were evaluated in bleomycin-induced pulmonary fibrosis and LPS-induced acute lung injury mouse models, utilizing lung-specific Ptges3 knockdown. AP covalently bound to Cys58 of prostaglandin E synthase 3 (Ptges3), an allosteric site distinct from its catalytic and Hsp90-binding regions. This binding inhibited Ptges3 enzymatic activity, reduced prostaglandin E2 (PGE 2 ) production, and disrupted the Ptges3-Hsp90 chaperone complex, leading to suppressed NF-κB signaling. Genetic knockdown of Ptges3 significantly attenuated the anti-inflammatory and antifibrotic effects of AP both in vitro and in vivo . Our findings establish Ptges3 as a critical functional target of AP. AP attenuates pulmonary fibrosis through a dual mechanism involving covalent inhibition of Ptges3 and disruption of the Ptges3-Hsp90-NF-κB axis, highlighting AP as a promising therapeutic agent and Ptges3 as a novel druggable target for IPF.
Zhang et al. (Fri,) studied this question.