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 pKa values, demonstrating that the dominant protonation state is retained under our NMR conditions and near physiological pH. Two orthogonal titration readouts─imino 1H 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.