De Tito, Stefano (2013) Molecular and macromolecular interactions of nucleic acids: an elaborate symphony. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Molecular and macromolecular interactions of nucleic acids: an elaborate symphony
Creators:
CreatorsEmail
De Tito, Stefanostefano.detito@unina.it
Date: 1 April 2013
Number of Pages: 209
Institution: Università degli Studi di Napoli Federico II
Department: Farmacia
Scuola di dottorato: Scienze farmaceutiche
Dottorato: Scienza del farmaco
Ciclo di dottorato: 25
Coordinatore del Corso di dottorato:
nomeemail
D'Auria, Maria Valeriamadauria@unina.it
Tutor:
nomeemail
Novellino, Ettorenovellin@unina.it
Date: 1 April 2013
Number of Pages: 209
Uncontrolled Keywords: nucleic acids; DNA; quadruplex
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 03 - Scienze chimiche > CHIM/02 - Chimica fisica
Area 03 - Scienze chimiche > CHIM/08 - Chimica farmaceutica
Date Deposited: 09 Apr 2013 11:49
Last Modified: 23 Jul 2014 08:53
URI: http://www.fedoa.unina.it/id/eprint/9308
DOI: 10.6092/UNINA/FEDOA/9308

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. Therefore, to achieve an enhanced knowledge on the structural and conformational requisites for quadruplex groove recognition, distamycin A, the only compound for which a pure groove binding has been proven, has been chemically modified. Isothermal titration calorimetry (ITC) and NMR techniques have been employed to characterize the interaction between a dicationic derivative of distamycin A (compound 1) and the [d(TGGGGT)]4 quadruplex structure. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity for the quadruplex structures. Further, structural and thermodynamic studies revealed that the absence of coulombic interactions inferred in compound 3, results in an unprecedented binding position in which both the groove and the 3’ end of the DNA are occupied. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)]4, we performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)]4 with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far. Among them, the promising derivative 6 (renamed 1a) was used as a seed for searching similar entities in several commercially available databases and NMR experiments allowed to identify a small focused library of structural analogues with G-quadruplex binding properties. By a back and forth approach, the structural features responsible for G-quadruplex groove recognition were delineated, while isothermal titration calorimetry (ITC) measurements allowed for the identification of chemotypes featuring a tighter binding than distamycin A. Differently from distamycin A, the best binders were also proved to be G-quadruplex selective over duplex. These results propelled the biological characterization of the new ligands demonstrating their ability to induce selective DNA damage at telomeric level and induction of apoptosis and senescence on tumor cells. Structural variations from the canonical Watson-Crick double helix have specific roles in many important cellular processes, such as DNA packaging, replication, transcription and recombination. These DNA structures are sequence-directed and constitute an alternative layer of the genetic code. Therefore, revealing new DNA structural motifs provides the molecular bases to elucidate novel functional mechanisms of cell and the way to interact with them. By combining advanced state-of-the-art computations and experiments, a study conducted also under the supervision of Prof. Bertini (CERM, Sesto Fiorentino) led to the identification of a new DNA structural motif, named “G-triplex”. G-triplex can be formed in guanine-rich regions of the genome and is characterized by the formation of G:G:G triad planes stabilized by an array of Hoogsteen-like hydrogen-bonds. This discovery further expands the structural complexity of the genome highlighting once more the high polymorphism of the DNA polymers. This is the first time that DNA is found to assume this kind of topology and the abundant presence of guanine-rich regions in the genome makes imperative to investigate its biological role in the near future. In a study conducted in Conte’s lab. (King’s College, London) the interactions between human LARP4 and poly-A15 RNA have been investigated. Human LARP4 (HsLARP4) is a cytoplasmic, polyribosome-associated protein involved in promoting mRNA translation and able to bind to oligo(A) RNA and the poly(A) binding protein (PABP). The RNA recognition is mediated by the La module, the conserved feature in all LARP families. However, human LARP4 is notably distinct from genuine La proteins and other LARPs for the lack of conservation of few amino acids that in human La were shown to be critical for 3’-UUUOH interaction, suggesting a divergent mode of RNA recognition. Here, five highly conserved residues in the La module of HsLARP4 were identified through sequence alignment with HsLa, and three of them (Q126, D139, M160) were mutated to Alanine through point mutation. ITC experiments revealed that the mutation of residues 126, 139 and 160 to Alanine in the La module of HsLARP4, decreased its binding affinity to poly-A15, hence, indicating that these residues of HsLARP4 play key roles in the interaction with RNA.

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