Fragment-based discovery, dynamics simulation and pharmacological study of 2-amino-pyrimidine derivative as HIPK2...

Chronic kidney disease (CKD) represents a major global public health challenge, pathologically characterized by renal fibrosis and frequently accompanied by inflammatory responses. Recent study indicates that homeodomain-interacting protein kinase 2 (HIPK2) is a key regulator of these fibrotic and inflammatory pathways. However, highly effective inhibitors targeting HIPK2 are currently lacking. In this study, we employed a fragment-based drug discovery (FBDD) strategy to develop a novel HIPK2 inhibitor, Hit 2c. Hit 2c demonstrated potent kinase inhibitory activity (IC₅₀ = 0.20 μM) and significant antiproliferative effects (NRK-49F IC₅₀ = 0.29 μM, induced by 10 ng/mL TGF-β). Molecular docking, molecular dynamics simulations, and free energy landscape analysis-revealed a stable binding mode between Hit 2c and HIPK2, in which a hydrogen bond formed with the key residue Lys288 serves as the central factor sustaining high binding affinity. Umbrella sampling further indicated that breaking this hydrogen bond requires a high dissociation energy barrier (3.22 kcal/mol). In vitro, Hit 2c inhibited HIPK2 downstream pathways (p53, TGF-β/Smad3, and NF-κB) and significantly downregulated fibrosis markers (Fn Ⅰ, Collagen Ⅰ, α-SMA) in NRK-49F cells induced by 10 ng/mL TGF-β and inflammatory cytokine IL-6 in HK-2 cells induced by 10 ng/mL TNF-α. In vivo, pharmacodynamic studies showed that oral administration of Hit 2c at 10 mg/kg attenuated renal injury and fibrosis in an adenine-induced mouse CKD model, comparable to the positive control dapagliflozin. Pharmacokinetic analysis revealed a half-life of 4.74 h, C_max of 426.35 ng/mL, AUC_0-∞ of 689.05 h ng/mL, suggesting relatively high clearance and low oral bioavailability (3.92 %). Liver microsome experiments suggested potential first-pass metabolism of Hit 2c. Collectively, Hit 2c represents a novel HIPK2 inhibitor scaffold with effective anti-fibrotic activity in vitro and in vivo, providing a lead compound for the development of HIPK2-targeted therapeutics.