Reframing Triazoloacridinone C-1305 as a G-Quadruplex Pharmacophore

What was once assigned as GGG intercalation can, in retrospect, be read as G-quadruplex recognition. Here, we reframe the clinically investigated triazoloacridinone C-1305 as a high-affinity ligand of the c-MYC promoter G4 model Pu22. UV–Vis speciation resolved four spectral forms and yielded three macroscopic p K a values, demonstrating that the dominant protonation state is retained under our NMR conditions and near physiological pH. Two orthogonal titration readouts─imino 1 H NMR and UV–Vis global analysis─converge on a well-defined 2:1 Pu22:C-1305 complex with submicromolar affinity and a distinctive ligand-induced “destabilize–refold” NMR signature. Variable-temperature NMR reveals pronounced thermal stabilization of Pu22 upon binding. Intermolecular NOEs together with 3D well-tempered metadynamics map a recognition mode dominated by end-stacking on both terminal G-tetrads, with flanking segments forming secondary pockets and enabling alternative bound substates. In cell assays, C-1305 shows low-micromolar cytotoxicity across multiple cancer cell lines while being markedly less toxic to normal cells, outperforming cisplatin in potency. These results reposition C-1305 from a putative GGG intercalator to a G4-targeting pharmacophore and outline a route to side-chain engineering for improved G4 selectivity.