A small molecule ACT004 targets four-and-a-half LIM domain protein 1 to disrupt TGF-β/Smad signaling and promote FHL1 degradation in renal fibrosis.

Renal fibrosis is a common endpoint of chronic kidney disease (CKD), yet effective targeted anti-fibrotic therapies remain limited. Ovatodiolide (OVA) has anti-fibrotic activity but poor drug-like properties, whereas ACT004, a synthetic OVA derivative, has improved water solubility and metabolic stability. However, whether ACT004 acts through the same mechanism as OVA or through a distinct target remains unclear. In this study, we evaluated the anti-fibrotic effects of ACT004 in unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) mouse models and investigated its underlying mechanism using RNA sequencing, HuProt human protein microarray, microscale thermophoresis, pull-down assays, cellular thermal shift assay, molecular docking, siRNA, mutant plasmids, co-immunoprecipitation, and pharmacological inhibition. ACT004 markedly attenuated renal fibrosis in UUO and UIRI mice and suppressed inflammatory signaling and Smad2/3 activation in vivo and in TGF-β1-stimulated renal tubular epithelial cells. Four-and-a-half LIM domain protein 1 (FHL1) was identified as a direct ACT004-binding target. ACT004 bound FHL1 at Lys102 and Thr128, and mutation of these residues weakened ACT004-FHL1 binding. FHL1 knockdown reduced TGF-β1-induced profibrotic activation, whereas ACT004 failed to further suppress fibrotic marker expression after FHL1 silencing. Mechanistically, ACT004 disrupted the FHL1-Smad2/3 interaction, inhibited Smad2/3 phosphorylation, and promoted ubiquitination-mediated degradation of FHL1. FHL1 overexpression aggravated UUO-induced renal fibrosis, whereas ACT004 alleviated FHL1-associated fibrotic injury in vivo. These findings identify FHL1 as a previously unrecognized pro-fibrotic scaffold in the kidney and position ACT004 as a next-generation anti-fibrotic candidate with a dual mechanism of action.