Screening of threading bis-intercalators binding to duplex DNA by electrospray ionization tandem mass spectrometry

Abstract

The DNA binding of novel threading bis-intercalators V1, trans-D1, and cis-C1, which contain two naphthalene diimide (NDI) intercalation units connected by a scaffold, was evaluated using electrospray ionization mass spectrometry (ESI-MS) and DNAse footprinting techniques. ESI-MS experiments confirmed that V1, the ligand containing the -Gly 3 -Lys-peptide scaffold, binds to a DNA duplex containing the 5'-GGTACC-3' specific binding site identified in previous NMR-based studies. The ligand formed complexes with a ligand/DNA binding stoichiometry of 1:1, even when there was excess ligand in solution. Trans-D1 and cis-C1 are new ligands containing a rigid spirotricyclic scaffold in the trans-and cis-orientations, respectively. Preliminary DNAse footprinting experiments identified possible specific binding sites of 5'-CAGTGA-5' for trans-D1 and 5'-GGTACC-3' for cis-C1. ESI-MS experiments revealed that both ligands bound to DNA duplexes containing the respective specific binding sequences, with cis-C1 exhibiting the most extensive binding based on a higher fraction of bound DNA value. Cis-C1 formed complexes with a dominant 1:1 binding stoichiometry, whereas trans-D1 was able to form 2:1 complexes at ligand/DNA molar ratios ≥ 1 which is suggestive of non-specific binding. Collisional activated dissociation (CAD) experiments indicate that DNA complexes containing V1, trans-D1, and cis-C1 have a unique fragmentation pathway, which was also observed for complexes containing the commercially available bisintercalator echinomycin, as a result of similar binding interactions, marked by intercalation in addition to hydrogen bonding by the scaffold with the DNA major or minor groove.

Evaluation of flavonoids binding to DNA duplexes by electrospray ionization mass spectrometry

“…Higher values of fraction of bound DNA indicate greater DNAbinding affinities. It has also been demonstrated in several studies that the relative binding affinities measured by ESI-MS were in fairly good agreement with the results obtained using solution-phase techniques [41][42][43]. Here, the relative binding affinities of the flavonoids toward duplex 1 are shown in Figure 2 as an example.…”

“…With an assumption that the ESI response factors of the free DNA and the drug/DNA complexes are identical, the relative intensities of the free and bound DNA in the mass spectra are expected to be proportional to their relative abundances in solution [34]. Therefore, the values of fraction of bound DNA can be used as a unique parameter to compare the relative binding affinities of drugs with DNA structures [41][42][43]. Higher values of fraction of bound DNA indicate greater DNAbinding affinities.…”

A study of the non-covalent interaction between flavonoids and DNA triplexes by electrospray ionization mass spectrometry

“…Electrospray ionization mass spectrometry (ESI-MS) has proved to be a powerful tool for drug discovery and examining drug/DNA complexes with advantages such as determination of precise stoichiometry, low sample consumption and short analysis time [24,25]. Many drug/DNA complexes have been extensively studied by ESI-MS, and the results have agreed well with those obtained using solution phase techniques [26][27][28][29][30][31][32][33][34][35][36][37]. The flavonoid/duplex DNA complexes were first investigated using ESI-MS in our laboratory [26].…”

“…Assuming that the addition of drug dose not changes the electrospray response factor of the DNA triplexes, the relative intensities of intact or bound DNA in the mass spectra are proportional to their relative abundance in the solution [35]. The value of fraction can be used to estimate the binding affinities and selectivities of drugs to DNA, and in many studies, results obtained using ESI-MS agreed well with those obtained in solution phase experiments [27,29]. The higher values of the fraction of bound DNA indicate greater DNA/drug binding affinities.…”

Intercalative pyrimido[4′,5′:4,5]thieno(2,3-b)quinolines induce apoptosis in leukemic cells: a comparative study of methoxy and morpholino substitution

“…Many chemotherapeutic agents against cancers are based on their interaction with cellular molecules like DNA or proteins [1]. DNA-binding molecules or intercalators represent a class of clinically useful drugs [2].…”