Virtual Screening, Molecular Dynamics, and Mechanism Study of Homeodomain-Interacting Protein Kinase 2 Inhibitor in Renal Fibroblasts

<b>Background/Objectives:</b> Homeodomain-interacting protein kinase 2 (HIPK2) is critically involved in the progression of renal fibrosis. This study aims to identify and characterize a novel HIPK2 inhibitor, CHR-6494, and investigate its therapeutic potential. <b>Methods:</b> Using structure-based virtual screening and molecular dynamics simulations, we identified CHR-6494 as a potent HIPK2 inhibitor with an IC₅₀ of 0.97 μM. The effects of CHR-6494 on the phosphorylation of p53 in Normal Rattus norvegicus kidney cells (NRK-49F) induced by transforming growth factor-β (TGF-β) were assessed, along with its impact on TGF-β signaling and downstream profibrotic markers. <b>Results:</b> CHR-6494 significantly reduces p53 phosphorylation induced by TGF-β and enhances the interaction between HIPK2 and seven in absentia 2 (SIAH2), facilitating HIPK2 degradation via proteasomal pathways. Both CHR-6494 and Abemaciclib inhibit NRK-49F cell proliferation and migration induced by TGF-β, suppressing TGF-β/Smad3 signaling and decreasing profibrotic markers such as Fibronectin I (FN-I) Collagen I and α-smooth muscle actin (α-SMA). Additionally, these compounds inhibit nuclear factor kappa-B (NF-κB) signaling and reduce inflammatory cytokine expression. <b>Conclusions:</b> The study highlights the dual functionality of HIPK2 kinase inhibitors like CHR-6494 and Abemaciclib as promising therapeutic candidates for renal fibrosis and inflammation. The findings provide new insights into HIPK2 inhibition mechanisms and suggest pathways for the design of novel HIPK2 inhibitors in the future.