DNA cleavage chemistry of pyridinium-based heterocyclic skipped aza-enediynes and targeting SV40 large T-antigen G-quadruplex DNA helicase activity by G-quadruplex interactive agents

Two diverse works regarding DNA-Drug Interaction are presented here. The first portion deals with covalent interactions between compounds that are derivatives of heterocyclic aza-enediynes and DNA (conventional Watson-Crick base paired double stranded DNA) and the second is related to non-covalent interactions of these compounds with G-quadruplex DNA. The aza-enediynes have been studied for their ability to undergo aza-variants of the Bergman and Myers cyclizations, and the potential role of the ensuing diradicals in DNA cleavage chemistry. The aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes has been recently reported. In the first part of the dissertation, the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-Npropargyl pyridine salts) are reported. Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. An optimized analog containing a p-methoxyphenyl substituent was prepared. Studies with radiolabeled DNA duplexes reveal that this analog generates nonselective frank DNA strand breaks, via deoxyribosyl 4'-hydrogen atom abstraction, and also leads to oxidation of DNA guanine bases. This is the first report of enediynelike radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization. The second part is based upon the growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for replication. A prototypical replicative helicase - SV40 large T-antigen (T-ag), a multifunctional protein with duplex DNA helicase activity is shown to also unwind G-quadruplex DNA structures. A series of G-quadruplex-interactive agents, particularly perylene diimide derivatives, is explored for inhibition of T-ag duplex and G-quadruplex DNA unwinding activities, and it is revealed that certain perylene diimides are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic Gquadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.Pharmac

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion.

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats

Simian virus 40 large T-antigen G-quadruplex DNA helicase inhibition by G-quadruplex DNA-interactive agents.

On the basis of growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for DNA replication, the G-quadruplex DNA unwinding ability of a prototypical replicative helicase, SV40 large T-antigen (T-ag), was investigated. Here, we demonstrate that this G-quadruplex helicase activity is robust and comparable to the duplex helicase activity of T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular G-quadruplexes, which are the presumed natural substrates for the G-quadruplex helicase activity of T-ag. A number of G-quadruplex-interactive agents as well as new perylene diimide (PDI) derivatives have been investigated as inhibitors of both the G-quadruplex and the duplex DNA helicase activities of T-ag. A unique subset of these G-quadruplex-interactive agents inhibits the G-quadruplex DNA unwinding activity of T-ag, relative to those reported to inhibit G-quadruplex DNA unwinding by RecQ-family helicases. We also find that certain PDIs are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic G-quadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication

Single-Molecule Manipulation of the Duplex Formation and Dissociation at the G-Quadruplex/i-Motif Site in the DNA Nanostructure

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing a G-quadruplex (GQ) forming and counterpart i-motif forming sequence in the DNA nanostructure. Sequential manipulation of DNA strands in the DNA frame was performed to prepare a topologically controlled GQ/i-motif dsDNA. Using strand displacement and the addition and removal of K+, the topologically controlled GQ/i-motif dsDNA in the DNA frame was obtained in high yield. The dsDNA was resolved into the single-stranded DNA, GQ, and i-motif by the addition of K+ and operation in acidic conditions. The dissociation of the dsDNA under the GQ and i-motif formation condition was monitored by high-speed atomic force microscopy. The results indicate that the dsDNA containing the GQ- and i-motif sequence is effectively dissolved when the duplex is helically loosened in the DNA nanoscaffold

Single-Molecule Manipulation of the Duplex Formation and Dissociation at the G‑Quadruplex/i-Motif Site in the DNA Nanostructure

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing a G-quadruplex (GQ) forming and counterpart i-motif forming sequence in the DNA nanostructure. Sequential manipulation of DNA strands in the DNA frame was performed to prepare a topologically controlled GQ/i-motif dsDNA. Using strand displacement and the addition and removal of K⁺, the topologically controlled GQ/i-motif dsDNA in the DNA frame was obtained in high yield. The dsDNA was resolved into the single-stranded DNA, GQ, and i-motif by the addition of K⁺ and operation in acidic conditions. The dissociation of the dsDNA under the GQ and i-motif formation condition was monitored by high-speed atomic force microscopy. The results indicate that the dsDNA containing the GQ- and i-motif sequence is effectively dissolved when the duplex is helically loosened in the DNA nanoscaffold

The Role of Substituent Effects in Tuning Metallophilic Interactions and Emission Energy of Bis-4-(2-pyridyl)-1,2,3-triazolatoplatinum(II) Complexes

The photoluminescence spectra of a series of 5-substituted pyridyl-1,2,3-triazolato PtII homoleptic complexes show weak emission tunability (ranging from λ=397-408 nm) in dilute (10-6 M) ethanolic solutions at the monomer level and strong tunability in concentrated solutions (10-4 M) and thin films (ranging from λ=487-625 nm) from dimeric excited states (excimers). The results of density functional calculations (PBE0) attribute this "turn-on" sensitivity and intensity in the excimer to strong Pt-Pt metallophilic interactions and a change in the excited-state character from singlet metal-to-ligand charge transfer (1MLCT) to singlet metal-metal-to-ligand charge transfer (1MMLCT) emissions in agreement with lifetime measurements. Turn-on tunability: A series of bis-4-(2-pyridyl)-1,2,3-triazolatoplatinum(II) complexes display variable emission tunability. At low concentration, the emission can be tuned only slightly by changing the nature of the substituent but at higher concentrations tunability is enhanced. This "turn-on" sensitivity in the excimeric emission is attributed to strong Pt-Pt metallophilic interactions and a change in the excited-state character