Drug repurposing-guided discovery of novel methyl quinoline-6-carboxylate RIPK3 inhibitors through structural optimization of Gefitinib.

Abstract

Receptor-interacting protein kinase 3 (RIPK3) is a central regulator of necroptosis and represents a promising therapeutic target for inflammatory and necroptosis-associated diseases. However, the discovery of potent and selective RIPK3 inhibitors remains challenging. In this study, a drug-repurposing strategy integrated with pharmacophore-based virtual screening was employed to identify novel RIPK3-targeting scaffolds from a library of FDA-approved drugs. Gefitinib was identified as a previously unrecognized RIPK3-binding compound with moderate anti-necroptotic activity and was subsequently selected as the starting scaffold for structural optimization. Guided by systematic structure-activity relationship studies, three rounds of medicinal chemistry optimization led to the discovery of compound 20 as the most promising analogue. Compound 20 exhibited potent anti-necroptotic activity with an EC50 value of 0.598 μM and enhanced RIPK3 binding affinity with a (Kd) value of 150 nM, representing a 9.3-fold improvement over Gefitinib. Kinase profiling against a panel of 80 kinases further demonstrated a favorable selectivity profile for RIPK3. Mechanistic studies showed that compound 20 suppressed RIPK3 activation, reduced downstream MLKL phosphorylation, and disrupted RIPK3-MLKL necrosome formation, thereby inhibiting necroptotic cell death. Molecular docking and molecular dynamics simulations indicated that compound 20 adopts a stable binding mode within the RIPK3 ATP-binding pocket, supported by persistent interactions with the key residues Met98 and Asp161. In a TNF-α/z-VAD-fmk-induced systemic inflammatory response syndrome mouse model, compound 20 significantly improved survival and alleviated multiple-organ injury. Experimental pharmacokinetic evaluation in mice revealed an oral bioavailability of 29.9% and an elimination half-life of 5.43 h following intragastric administration, supporting sustained systemic exposure. Overall, this study identifies Gefitinib as a novel RIPK3-targeting scaffold and demonstrates that structure-guided optimization of an EGFR inhibitor can yield potent and selective RIPK3-directed anti-necroptotic agents. Compound 20 represents a promising lead for further optimization toward the treatment of necroptosis-associated inflammatory diseases.