Browsing by Author "Carrilho, Rui Pedro Abrantes"
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- Molecular Modelling of a novel G-quadruplex structure and its interaction with ligandsPublication . Carrilho, Rui Pedro Abrantes; Cruz, Carla Patrícia Alves Freire Madeira; Sousa, Fani Pereira deDNA can exist under many different forms. Lately, G-quadruplexes, which are one example of the non-canonical DNA forms, have been getting a lot of attention due to the role they play in certain biological processes and as potential targets for therapeutic interventions. For example, these structures can exist in certain parts of the telomeres, structures responsible for cell replication. In cancer cells, if the enzyme telomerase could be inhibited, by inducing the formation of a G-quadruplex structure in guanine-rich telomere sequences, the spread of cancer cells would cease. For this and other reasons, it becomes important to be able to induce the formation of G-quadruplex structures and/or stabilize them, and one of the ways of doing so consists of targeting these sequences with ligands that have good affinity to G-quadruplex structures. However, few G-quadruplex ligands demonstrated the needed properties to fulfill the clinical needs, and further efforts to determine which would be better suited to target any particular sequence are needed. This work aimed at comparing the affinity to the pre-miR-149 G-quadruplex structure of seven promising ligands found in the literature, through the latest techniques fit for that purpose. The seven ligands tested were: [16]phenN2, [32]phen2N4, phen-DC3, pyridostatin, acridine orange derivatives C8 and C8-NH2 and L-arginine. Firstly, they underwent computational tests, with the molecular structure of the quadruplex and the ligand being simulated, and their optimal binding site and conformation found. Their binding energies were compared, and they underwent molecular dynamics runs to simulate their behavior in an environment with solvent, followed by another binding energy comparison. The trend obtained in order of decreasing binding affinity was: pyridostatin > [32]phen2N4 > [16]phenN2 > Phen-DC3 > L-arginine > C8 > C8-NH2. Biophysical techniques were then performed, to determine the binding affinities experimentally. First, circular dichroism spectroscopy and melting studies (performed on four ligands) established the following trend: C8 > pyridostatin > C8-NH2 > [16]phenN2. Fluorescence spectroscopy titration (performed on three) revealed a similar trend: C8 > C8-NH2 > [16]phenN2. Lastly, affinity chromatography experiments were held to test how other DNA sequences would bind to C8-NH2. The results revealed that the ligand has better binding affinity with parallel quadruplexes over antiparallel ones, and poor binding with a duplex sequence. Overall, the best ligands identified for binding to the G-quadruplex structure were the acridine orange derivatives C8 and C8-NH2, and pyridostatin. These three ligands should be considered prime candidates for further research in this area.