Binding Does Not Mean Stabilizing: High-Affinity Porphyrins Exhibit Opposite Effects on G-Quadruplex Stability

Binding affinity is often used as a predictor of G-quadruplex (G4) stabilization. Here, we show that ligand binding and quadruplex stabilization can be thermodynamically decoupled. Two closely related tetracationic porphyrins bind the human telomeric G4 with comparable affinities approaching 106 M–1 but induce opposite thermal responses. TMPyP4 stabilizes the quadruplex (+9 °C), whereas PL7 destabilizes it (−6 °C) despite a similar binding free energy. Thermodynamic analysis reveals distinct binding mechanisms, with TMPyP4 association driven primarily by favorable enthalpy and PL7 binding dominated by entropy. Temperature-dependent circular dichroism measurements indicate that PL7 promotes redistribution within the telomeric G4 conformational ensemble rather than stabilization of the native hybrid fold. These findings demonstrate that high affinity does not necessarily imply G4 stabilization and establish affinity and thermal stabilization as independent descriptors of ligand–quadruplex interactions.