Dna Secondary Structures As Targets For Anticancer Drugs

DNA secondary structures containing stretches of single stranded DNA are present in the human genome and are involved in the regulation of crucial processes such as transcription. Hairpins or cruciforms are the potential recognition sites for binding of transcription factors. Of equal importance are telomeres, the DNAprotein complexes marking the ends of chromosomes. Facile interconversion between double and single stranded DNA and G-quadruplex at physiological conditions renders these secondary DNA structures attractive candidates for biological signaling molecules and consequently, potential targets for anticancer agents.

4.1. Targeting DNA Quadruplexes

Telomeres are DNA protein complexes at the ends of eukaryotic chromosomes that protect the linear DNA ends from erosion over multiple replication cycles and also from being recognized as double strand breaks and subsequent repair by exonucle-olytic trimming and end-to-end fusion. Chromosomal telomeres contain 3' G-rich overhang of 150—200 bp that forms a G-quartet structure. It is in the form of stacked tetrads of guanines in a cyclic Hoogsteen hydrogen bonding arrangement. G-quartets can be stabilized by sodium and potassium ions and this stabilization can inhibit telomerase activity. A number of small molecules have been identified that interact with G-quartets. For instance, 2,6-diamidoanthraquinone BSU1051 has been found to interact with and stabilize the G-quartet, and thereby inhibit telomerase activity (Sun et al., 1997). A 3,6,9-trisubstituted acridine is also a potent inhibitor of telomerase. Apart from these, certain cationic porphyrins such as TMPyP4 are another class of agents that bind to G-tetrads by interactive stacking. TMPyP4 has selectivity for intermolecular G-quadruplex structures (Liu et al., 2005). Telomes-

tatin is a natural G-quadruplex intercalating agent, which holds greater promise compared to previously studied G-quadruplex targeted molecules. On treatment of multiple myeloma cells with Telomestatin, inhibition of telomerase activity occurs along with reduction in telomere length followed by cell growth inhibition (Shammas et al., 2004). A third class of G-tetrad interacting compounds comprises of perylenetetracarboxylic diimide PIPER which shows binding characteristics, similar to the porphyrins. These compounds not only bind to G-quadruplexes, but also induce their formation in cells (Han et al., 1999).

Apart from telomeres, G-quadruplexes are also present in the upstream promoter regions of certain oncogenes. G-quadruplex targeted molecules may interact at these sites as well. In fact, the cationic prophyrin, TMPyP4 and the core modified expanded prophyrin analogue 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP) have been found to cause repression of transcriptional activation of c-MYC in cells by G-quadruplex stabilization (Seenisamy et al., 2005).

4.2. Targeting Hairpins and Holliday Junctions

Hairpins, cruciforms or single strand DNA containing secondary structures play an important role in the regulation of transcription. A molecule that can selectively bind to hairpins has the potential to block transcription by interfering with protein recognition of that specific site. The transcription inhibitor Actinomycin D and its analogues bind nearly 10 fold more tightly to the hairpin conformation formed from the single stranded DNA 5'-A7TAGT4A3TAT7-3' than to the same strand in fully duplexed form.

4.3. Targeting DNA Triplexes

Specific targeting of triplex DNA structures is one of the most important strategies of 'antigene' based chemotherapy (Jenkins, 2000). The main aim is to target individual gene sequences at the DNA duplex level to modulate their expression or interactions with DNA binding proteins or to interfere with the vital template processes. A genomic DNA duplex is targeted using either a triplex forming oligonucleotide (TFO) or peptide nucleic acid analogue (PNA) to produce local DNA triplex structures that can inhibit the transcription of specific genes. The specificity of these triple helical structures stems from the Z—X ■ Y base triplets formed by Hoogsteen or reverse Hoogsteen hydrogen bonding arrangements involving pyrimidine or purine bases (Z) in the third strand and the purine strand (X) of the host DNA duplex. The triple helical structures formed have low thermal and thermodynamic stability and this poses a problem for effective targeting. So DNA triplex-targeted drugs mainly aim at stabilization of DNA triplexes.

Significant increase in triplex stability can be achieved by using intercalating agents, either by conjugate attachment to the third strand TFO or as a separate adjunct ligand, if binding of this residue can preferentially stabilize a triple stranded DNA. Various fused and unfused heterocycles have been studied. They include naphthylquinoline, BePI (benzo[e]pyridoindole), imidazothioxan-thone, acridine derivatives, coralyne, and the phenothiazinium dye, methylene blue. These molecules have (i) an extended planar aromatic system to maximize ^-overlap with successive base triplet planes, (ii) a cationic charge on the intercalated chromophore and (iii) a pendant side-chain terminating in an amine residue that can be protonated. Due to these characteristics, the compound is easily delivered to the target DNA and it anchors the bound ligand through interaction with the grooves or the anionic phosphodiester backbone.

Efforts have also been made to design groove directed drugs for triplex stabilization. Ideally, these agents should have little or no inherent affinity for the underlying duplex in order to prevent intergroove cross talk and consequent binding induced displacement of the third strand. However, most of the compounds studied are mainly A/T specific with high affinity for DNA duplexes rather than DNA triplexes.

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