Evaluation of metal-mediated DNA binding of benzoxazole ligands by electrospray ionization mass spectrometry

Abstract

The binding of a series of benzoxazole analogs with different amide-and ester-linked side chains to duplex DNA in the absence and presence of divalent metal cations is examined. All ligands were found to form complexes with Ni 2ϩ , Cu 2ϩ , and Zn 2ϩ , with 2:1 ligand/metal cation binding stoichiometries dominating for ligands containing shorter side chains (2, 6, 7, and 8), while 1:1 complexes were the most abundant for ligands with long side chains (9, 10, and 11). Ligand binding with duplex DNA in the absence of metal cations was assessed, and the long side-chain ligands were found to form low abundance complexes with 1:1 ligand/ DNA binding stoichiometries. The ligands with the shorter side chains only formed DNA complexes in the presence of metal cations, most notably for 7 and 8 binding to DNA in the presence of Cu 2ϩ . The binding of long side-chain ligands was enhanced by Cu . As a topoisomerase II inhibitor, one of the unique properties of UK-1 is its ability to bind biologically important divalent metal cations [3] and its metalmediated DNA binding [3,7]. While UK-1 has demonstrated cytotoxicity against a number of cell lines, it does not inhibit the growth of bacteria, yeast, or fungi [1,8], making the mechanism of this selective cytotoxic activity and the metal binding properties of UK-1 and new analogs of great interest.In a recent study, we used electrospray ionization mass spectrometry (ESI-MS) in conjunction with cytotoxicity assays to examine several simple analogs of UK-1 to explore the metal ion binding requirements of these compounds, assess metal-mediated DNA binding and evaluate anticancer and antibacterial activity [6]. Interestingly, the only ligand that exhibited anticancer activity, 2 (Figure 1), was also the only metal-mediated DNA binder with a preference for Ni 2ϩ as determined by ESI-MS experiments. Two other compounds, 4 and 5, formed complexes with DNA in a nonspecific, nonmetalmediated manner, and showed antibacterial but not cytotoxic behavior. These results suggested a correlation between metal-mediated binding and anticancer activity of the compounds. To improve upon the solubility of the benzoxazoles in aqueous solutions and to allow further examination of the metal-mediated DNA binding and anticancer activity, a series of analogs of 2 have been synthesized with different ester and amidelinked side chains (Figure 1) [9].In the present study, ESI-MS was used to screen the binding of the new ligands to a series of divalent metals including Mg 2ϩ , Ni 2ϩ , Cu 2ϩ , and Zn 2ϩ . Ligand binding to duplex DNA in the presence and absence of metal ions was also assessed. ESI-MS has been shown to be a useful tool for the analysis of noncovalent ligand/DNA

Synthesis, metal ion binding, and biological evaluation of new anticancer 2-(2′-hydroxyphenyl)benzoxazole analogs of UK-1

“…Despite the pronounced effect of 4-position substituents on Mg 2+ ion binding affinity in the 2-(2'-hydroxyphenyl)benzoxazole series, there are only relatively subtle variations in cytotoxicity. Our recent ESI-MS investigation of the metal ion binding of these compounds failed to demonstrate formation of Mg 2+ complexes in aqueous buffers, although complexes were observed in methanol [23]. These and previous ESI-MS studies [10] failed to demonstrate formation of ligand-Mg 2+ -DNA complexes with UK-1 or these analogs, although ligand-Cu 2+ -DNA complexes were readily observed.…”

“…The cytotoxic analogs 1, 12-14, and 17 all demonstrate enhanced DNA binding by ESI-MS in the presence of Cu 2+ or Ni 2+ ; whereas, the relatively non-cytotoxic analogs 15 and 16 do not [23]. Even the 7-substituted benzoxazole 3 forms ligand-metal ion-DNA complexes, although these complexes are formed in much lower abundance when compared to those formed in the presence of 1 [10].…”

Evaluation of DNA/Ligand Interactions by Electrospray Ionization Mass Spectrometry

“…ESI-MS has proven to be useful for exploring DNA/ligand complexation in this context (23)(24)(25). One of the original studies reported the use of ESI-MS to examine the metal-mediated binding of chromomycin A 3 and mithramycin, two antitumor agents (23).…”

“…The metal-mediated binding of a series of benzoxazole and benzimidazole ligands (Figure 3) has also been investigated. In these studies (24,25), solutions containing DNA, one ligand, and either one divalent metal or no metal were analyzed by ESI-MS. These ligands were developed as analogs of UK-1, a natural product isolated from Streptomyces that possesses strong anticancer activity presumed to arise from its metal-mediated catalytic inhibition of human topoisomerase II.…”

“…The observed poly(dGdC) preference of the ligands was thought to stem from (a) enhanced hydrogen bonding between the intercalators and functional groups in the major and minor grooves of G/C-rich sequences as well as (b) more favorable electrostatic interactions between the imide carbonyls of the naphthalene diimide groups and the N 2 amino group on G/C base pairs. The sequence selectivities of cis-C1 T m (°C ) 25 10…”