Compounds are Highly Selective for Quadruplex-DNA over Duplex-DNA

With the abundance of duplex DNA in the genome, targeting relatively few non-duplex structures such as quadruplexes becomes a complex task. Non-selective binding to secondary sites might result in deleterious effects and cytotoxicity, therefore, structure and sequence selectivity is of utmost importance to achieve desired results. In order to determine the selectivity of DB compounds for human telomeric quadruplex sequence over duplex DNA sequences, thermal melting studies were performed with telomeric quadruplex DNA (Tel22: Materials and Methods, 4.2.1) and two different duplex sequences (AATT, GC20: Materials and Methods, 4.2.1). The AT-rich duplex sequence was chosen since small molecules show a preferential binding towards sequences with a narrow minor groove, and a GC-rich duplex sequence was chosen to mimic the wider grooves of quadruplex DNA and, also, DB293, as aforementioned, has been shown to cooperatively bind as a stacked dimer in GC- containing sequences. This would also eliminate any bias towards any particular groove width for the tested compounds. Melting curves were obtained for quadruplex sequences up to ratios as high as 6:1 compound per quadruplex and ratios as high as 4:1 for duplex sequences. The ∆Tm values for quadruplex (2:1, 4:1 ratios) and duplex sequences (2:1 ratio) are reported in Table 4.1; ratios higher than the listed range resulted in the signification complex aggregation and produced inconsistent melting curves. This high stoichiometry was used because, as aforementioned, the recently solved NMR structure of distamycin bound to an intermolecular

quadruplex structure shows that up to four distamycin molecules are bound in the opposite grooves of the quadruplex. Therefore, it is a likely possibility that relatively small-sized molecules that mimic distamycin can bind to quadruplex DNA with a high stoichiometry. The melting temperature of Tel22 in 100 mM K+ is 48.8 °C. DB832, the paradigm compound, showed a ∆Tm of 19.1 °C at 2:1 and an impressive 26.1 °C at 4:1 ratios indicating signification stabilization of the quadruplex conformation. Also, with the two duplex sequences, Dickerson and GC20, DB832 shows very little increase in the Tm (3 °C and 3.7 °C respectively at 2:1 ratio) suggesting high selectivity over duplex DNA. Interestingly, DB1093, a more linear structural isomer of DB832, showed almost no change in ∆Tm at 2:1 ratio and a small increase (5 °C) at 4:1 ratio highlighting the importance of the neighboring furan ring systems for effective recognition of quadruplex architecture. DB1093 also exhibited very little change in ∆Tm for duplex sequences (Table 4.1) suggesting that the compound is too linear to interact with duplex DNA minor groove. The substitution of the two furan rings with thiophenes, DB1450, results in very similar quadruplex stabilization (2:1-17.6 °C, 4:1-24.2 °C) as DB832 but, unfortunately, this modification decreases the specificity, with a ΔTm of almost 9 °C, for

the duplex sequences. Affinity for duplex sequences decreases by an impressive 6°C, relative to DB1450, when the terminal furan is replaced by a thiophene (DB1438), while maintaining strong quadruplex stabilization potential (4:1-25.5 °C). Interestingly, the substitution of the central furan by a thiophene, DB1463, significantly increases the binding to AATT sequence (8.5 °C), but not the GC20 sequence (3.4 °C), suggesting the effect of compound curvature on recognizing different groove dimensions. However, the quadruplex stabilization for DB1463 is not very different (2:1-17.3 °C, 4:1-23.1 °C) from what was observed for DB832. The addition of a freely rotatable triple bond between the phenyl and furan ring systems, DB1694, also significantly decreases the affinity for duplex sequences (AATT-1.0 °C, GC20-2.1 °C) while maintaining good stabilization properties with quadruplex DNA. This has been previously

observed in the case of diaryl amides where the addition of the acetylene group has increased quadruplex recognition potential [29]_{. The nitrogen substitution on the phenyl ring of DB832}

exhibited enhanced stabilization of the quadruplex arrangement when it is placed in the meta position (DB1693) but slightly decreased affinity when it is in ortho (DB934). The conversion of amidine groups to imidazolines (DB1972 and DB2037) yields a more planar arrangement while still maintaining a dicationic charge system. These modifications provide an increased stacking surface available for ligand interaction with the terminal tetrads, and might have higher quadruplex recognition potential. The imidazoline analogs, however, exhibited generally lower ∆Tm than amidine counterparts. The oxazole derivative of DB832, DB1999, and the addition of a methyl group on terminal furan, DB1949, also had high quadruplex stabilization properties while and very little affinity for duplex sequences under low salt conditions. The reference compound, distamycin, recently proposed as a G-quadruplex groove binder with the four-stranded d(TG4T) at NMR concentrations, did not show any ΔTm with the

human telomere quadruplex sequence. As it is well known, however, that the polyamide is a strong binder to AT sequences in duplex DNA, distamycin is the only compound of this group that has reverse selectivity for duplex over quadruplex. Finally, RHPS4, a well-studied quadruplex stabilizing compound that has been reported to bind by π-π interactions with the external tetrad, shows melting values lower than to DB832 in terms of stabilization properties and selectivity for quadruplex over duplex DNA. The quadruplex stabilization potential observed for some of the DB compounds ranks among the strongest for quadruplex-interacting small molecules reported so far. It is highly apparent that the ligands are significantly stabilizing the quadruplex conformation, while maintaining a low degree of selectivity for duplex sequences. Therefore, these compounds make ideal candidates for further study as highly selective quadruplex interacting agents.

4.3.2 DB Compounds Bind to the Human Telomeric DNA with Multiple Binding Modes