Structure-guided discovery of a dual JAK2/RAGE modulator reveals a computationally driven strategy for targeting convergent oncogenic signaling in renal cell carcinoma.

Renal cell carcinoma (RCC) is driven by convergent inflammatory and proliferative signaling pathways. RAGE-mediated NF-κB activation and JAK2-STAT3 signaling cooperate to amplify cytokine production and oncogenic progression. Targeting these interconnected pathways represents a rational therapeutic strategy. High-throughput virtual screening of ChemBridge libraries, 100 ns molecular dynamics simulations, and MM/PBSA analysis were performed. The lead compound RJS6641 was evaluated using biochemical assays (HTRF JAK2 kinase assay, differential scanning fluorimetry), a cell-free AGE-RAGE binding ELISA, and functional assays in RCC cell lines, including viability, reporter, cytokine, apoptosis, and migration analyses. RJS6641 showed stable binding to JAK2 (protein RMSD ≈0.28 nm; ligand RMSD 0.15-0.20 nm) and RAGE (protein RMSD 0.50-1.50 nm; ligand RMSD 0.30-0.35 nm). It inhibited JAK2 in an ATP-competitive manner (IC₅₀ = 60.23 nM; ruxolitinib IC₅₀ = 1.69 nM), with ATP-dependent shifts (35.04 → 74.09 → 305.6 nM) and thermal stabilization (ΔTm +1.50 °C). AGE-RAGE binding was disrupted (IC₅₀ = 118.2 nM). RJS6641 reduced RCC viability (GI₅₀: 799.8-2021 nM) while sparing HK-2 cells (GI₅₀ 12,816 nM; 6-16× difference). It suppressed STAT3 (IC₅₀ 412.3-813.2 nM) and NF-κB (IC₅₀ 435.7-1031 nM) signaling, reduced IL-6/IL-8 secretion, induced caspase-3/7 activation (EC₅₀ 662.4-862.9 nM), and inhibited migration (up to 83%). Collectively, RJS6641 functions as a JAK2 inhibitor with additional modulation of RAGE-associated signaling, leading to coordinated suppression of STAT3/NF-κB pathways and anti-tumor effects in RCC, supporting further in vivo and selectivity evaluation.