Novel pyrimidopyrimidine derivatives for inhibition of cellular proliferation and motility induced by h-prune in breast cancer.

The human (h)-prune protein is a member of the DHH protein superfamily and it has a cAMP phosphodiesterase activity. Its overexpression in breast, colorectal and gastric cancers correlates with depth of invasion and a high degree of lymph-node metastasis. One mechanism by which h-prune stimulates cell motility and metastasis processes is through its phosphodiesterase activity, which can be suppressed by dipyridamole, a pyrimido[5,4-d]pyrimidine analogue. To obtain new and more potent agents that have high specificity towards inhibition of this h-prune activity, we followed structure-activity-relationship methodologies starting from dipyridamole and synthesised eight new pyrimido-pyrimidine derivatives. We analysed these newly generated compounds for specificity towards h-prune activities in vitro in cellular models using scintillation proximity assay for cAMP-PDE activity, cell index in cell proliferation assays and transwell methodology for two-dimensional cell migration in a top-down strategy of selection. Our findings show that two pyrimido[5,4-d]pyrimidine compounds are more effective than dipyridamole in two highly metastatic cellular models of breast cancer in vitro. Future studies will assess their therapeutic effectiveness against breast and other cancers where there is over-expression of h-prune, and in ad-hoc, proof of concept, animal models.

Structural investigations on the anti-HIV G-quadruplex-forming oligonucleotide TGGGAG and its analogues: evidence for the presence of an A-tetrad.

Several anti-HIV aptamers adopt DNA quadruplex structures. Among these, "Hotoda's aptamer" (base sequence TGGGAG) was one of the first to be discovered. Although it has been the topic of some recent research, no detailed structural investigations have been reported. Here we report structural investigations on this aptamer and analogues with related sequences, by using UV, CD, and NMR spectroscopy as well as electrophoretic techniques. The addition of a 3'-end thymine has allowed us to obtain a single, investigable quadruplex structure. Data clearly point to the presence of an A-tetrad. Furthermore, the effects of the incorporation of an 8-methyl-2'-deoxyguanosine at the 5'-end of the G-run were investigated.

The abasic site lesions in the human telomeric sequence d[TA(G(3)T(2)A)(3)G(3)]: a thermodynamic point of view.

BACKGROUND: The abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes. METHODS: In this paper, we report investigations, based on calorimetric, UV, CD and electrophoretic techniques, on 12 oligodeoxynucleotides analogues of the quadruplex forming human telomere sequence d[TA(G(3)T(2)A)(3)G(3)], in which each guanine has been replaced, one at a time, by an abasic site mimic. RESULTS: Although all data show that the modified sequences preserve their ability to form quadruplex structures, the thermodynamic parameters clearly indicate that the presence of an abasic site decreases their thermal stability compared to the parent unmodified sequence, particularly if the replacement concerns one of the guanosines involved in the formation of the central G-tetrad. CONCLUSIONS: The collected data indicate that the effects of the presence of abasic site lesions in telomeric quadruplex structures are site-specific. The most dramatic consequences come out when this lesion involves a guanosine in the centre of a G-run. GENERAL SIGNIFICANCE: Abasic sites, by facilitating the G-quadruplex disruption, could favour the formation of the telomerase primer. Furthermore they could have implications in the pharmacological approach targeting telomere.

The insertion of two 8-methyl-2'-deoxyguanosine residues in tetramolecular quadruplex structures: trying to orientate the strands.

In this article, we report a structural study, based on NMR and CD spectroscopies, and molecular modelling of all possible d(TG(3)T) and d(TG(4)T) analogues containing two 8-methyl-2'-deoxyguanosine residues (M). Particularly, the potential ability of these modified residues to orientate the strands and then to affect the folding topology of tetramolecular quadruplex structures has been investigated. Oligodeoxynucleotides (ODNs) TMMGT (T12) and TMMGGT (F12) form parallel tetramolecular quadruplexes, characterized by an all-syn M-tetrad at the 5'-side stacked to all-anti M- and G-tetrads. ODNs TMGMT (T13) and TMGGMT (F14) form parallel tetramolecular quadruplexes, in which an all-anti G core is sandwiched between two all-syn M-tetrads at the 5'- and the 3'-side. Notably, the quadruplex formed by T13 corresponds to an unprecedented structure in which the syn residues exceed in number the anti ones. Conversely, ODN TGMGMT (F24) adopts a parallel arrangement in which all-anti G-tetrads alternate with all-syn M-tetrads. Most importantly, all data strongly suggest that ODN TMGMGT (F13) forms an unprecedented anti-parallel tetramolecular quadruplex in which G and M residues adopt anti and syn glycosidic conformations, respectively. This article opens up new understandings and perspectives about the intricate relationship between the quadruplex strands orientation and the glycosidic conformation of the residues.

Unprecedented right- and left-handed quadruplex structures formed by heterochiral oligodeoxyribonucleotides.

CD and NMR studies on heterochiral oligodeoxynucleotides (d/l-ODNs) forming quadruplex structures are reported. Heterochiral ODNs, based on sequence TGGGGT, are able to form stable either right- or left-handed quadruplexes depending on d/l ratio and residues position. Results suggest that the 3'-end and the core of the G-run are more important than the 5'-end in determining the quadruplex handness. Particularly, oligonucleotide T(D)G(D)G(L)G(L)G(D)T(D) (L34) at low temperatures forms a well-defined left-handed quadruplex, notwithstanding it is mostly composed by natural d residues. This structure is characterized by three all-anti G-tetrads and one all-syn G-tetrad.

Effects of 8-methylguanine on structure, stability and kinetics of formation of tetramolecular quadruplexes.

Tetramolecular G-quadruplexes result from the association of four guanine-rich strands. Modification of the backbone strand or the guanine bases of the oligonucleotide may improve stability or introduce new functionalities. In this regard, the 8 position of a guanosine is particularly suitable for introduction of modifications since as it is positioned in the groove of the quadruplex structure. Modifications at this position should not interfere with structural assembly as would changes at Watson-Crick and Hoogsteen sites. In this study, we investigated the effect of an 8-methyl-2'-deoxyguanosine residue (M) on the structure and stability of tetramolecular parallel G-quadruplexes. In some cases, the presence of this residue resulted in the formation of unusual quadruplex structures containing all-syn tetrads. Furthermore, the modified nucleoside M at the 5'-end of the sequence accelerated quadruplex formation by 15-fold or more relative to the unmodified oligonucleotide, which makes this nucleobase an attractive replacement for guanine in the context of tetramolecular parallel quadruplexes.

Effects of abasic sites on structural, thermodynamic and kinetic properties of quadruplex structures.

Abasic sites represent the most frequent lesion in DNA. Since several events generating abasic sites concern guanines, this damage is particularly important in quadruplex forming G-rich sequences, many of which are believed to be involved in several biological roles. However, the effects of abasic sites in sequences forming quadruplexes have been poorly studied. Here, we investigated the effects of abasic site mimics on structural, thermodynamic and kinetic properties of parallel quadruplexes. Investigation concerned five oligodeoxynucleotides based on the sequence d(TGGGGGT), in which all guanines have been replaced, one at a time, by an abasic site mimic (dS). All sequences preserve their ability to form quadruplexes; however, both spectroscopic and kinetic experiments point to sequence-dependent different effects on the structural flexibility and stability. Sequences d(TSGGGGT) and d(TGGGGST) form quite stable quadruplexes; however, for the other sequences, the introduction of the dS in proximity of the 3'-end decreases the stability more considerably than the 5'-end. Noteworthy, sequence d(TGSGGGT) forms a quadruplex where dS does not hamper the stacking between the G-tetrads adjacent to it. These results strongly argue for the central role of apurinic/apyrimidinic site damages and they encourage the production of further studies to better delineate the consequences of their presence in the biological relevant regions of the genome.