The nature of the binding mode and stoichiometry of the TMPyP4 cationic porphyrin to G-quadruplex structures continues to be controversial, with no consensus model to date, especially for intramolecular G-quadruplexes from human telomeric sequences. Those sequences possess intricate polymorphism in solution that appears to be reduced under molecular crowding conditions in which the parallel structure appears to be the most populated one. We have performed a systematic study, in dilute solution and under molecular crowding conditions, of the binding reactions between TMPyP4 and four G-quadruplexes formed by different truncations of human telomeric DNA, with 5'- or 3'-flanking bases, using isothermal titration calorimetry and circular dichroism. The results clearly indicate that all of these G-quadruplexes are able to bind up to four TMPyP4 molecules. CD studies show that interaction with TMPyP4 promotes the conversion of the hybrid structures to an antiparallel conformation in dilute solution, while under molecular crowding conditions the interaction does not promote any conformational change. ITC reveals in both cases that the binding process comprises two sequential events, a first in which one molecule of TMPyP4 interacts with the quadruplex structures and a second in which three other molecules bind to the structures. The selectivity of TMPyP4 for the quadruplex relative to duplex DNA was also investigated under molecular crowding conditions showing that TMPyP4 has enhanced selectivity for quadruplex DNA compared to the duplex structure. This finding reinforces the potential applications of TMPyP4.

No abstract

Targeting of DNA secondary structures, such as G-quadruplexes, is now considered an appealing opportunity for drug intervention in anticancer therapy. So far, efforts made in the discovery of chemotypes able to target G-quadruplexes mainly succeeded in the identification of a number of polyaromatic compounds featuring end-stacking binding properties. Against this general trend, we were persuaded that the G-quadruplex grooves can recognize molecular entities with better drug-like and selectivity properties. From this idea, a set of small molecules was identified and the structural features responsible for G-quadruplex recognition were delineated. These compounds were demonstrated to have enhanced affinity and selectivity for the G-quadruplex over the duplex structure. Their ability to induce selective DNA damage at telomeric level and to induction of apoptosis and senescence on tumor cells is herein experimentally proven.