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Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

ABSTRACT: Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

SUBMITTER: Ranjan N

PROVIDER: S-EPMC4048829 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

Ranjan Nihar N Kumar Sunil S Watkins Derrick D Wang Deyun D Appella Daniel H DH Arya Dev P DP

Bioorganic & medicinal chemistry letters 20130814 20

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturati ...[more]

PMID: 24012122

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Project description:G-quadruplexes have been characterized as structures of vital importance in the cellular functioning of several life forms. They have subsequently been established to serve as a therapeutic target of several diseases including cancer, HIV, tuberculosis and malaria. In this paper, we report the binding of aminosugar-intercalator conjugates with a well-studied anti-parallel G-quadruplex derived from Oxytricha Nova G-quadruplex DNA. Of the four neomycin-intercalator conjugates studied with varying surface areas, BQQ-neomycin conjugate displayed the best binding to this DNA G-quadruplex structure with an association constant of K a = (1.01 ±0.03) × 107 M-1 which is nearly 100-fold higher than the binding of neomycin to this quadruplex. The binding of BQQ-neomycin displays a binding stoichiometry of 1:1 indicating the presence of a single and unique binding site for this G-quadruplex. In contrast, the BQQ-neomycin displays very weak binding to the bacterial A-site rRNA sequence showing that BQQ-does not enhance the neomycin binding to its natural target, the bacterial rRNA A-site. The BQQ-neomycin conjugate is prone to aggregation even at low micromolar concentrations (4 μM) leading to some ambiguities in the analysis of thermal denaturation profiles. Circular dichroism experiments showed that binding of BQQ-neomycin conjugate causes some structural changes in the quadruplex while still maintaining the overall anti-parallel structure. Finally, the molecular docking experiments suggest that molecular surface plays an important role in the recognition of a second site on the G-quadruplex. Overall, these results show that molecules with more than one binding moieties can be made to specifically recognize G-quadruplexes with high affinities. The dual binding molecules comprise of quadruplex groove binding and intercalator units, and the molecular surface of the intercalator plays an important part in enhancing binding interaction to the G-quadruplex structure.

Project description:Thermodynamic studies on the interactions between intercalator-neomycin conjugates and a DNA polynucleotide triplex [poly(dA).2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalene diimide-neomycin (3) and anthraquinone-neomycin (4) conjugates were synthesized and used together with two other analogues, previously synthesized pyrene-neomycin (1) and BQQ-neomycin (2) conjugates, in our investigations. A combination of experiments, including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC), revealed that all four conjugates (1-4) stabilized poly(dA).2poly(dT) much more than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (T(m3-->2)) at which poly(dA).2poly(dT) dissociated into poly(dA).poly(dT) and poly(dT) increased dramatically (>12 degrees C) in the presence of intercalator-neomycin conjugates (1-4) even at a very low concentration (2 muM). In contrast to intercalator-neomycin conjugates, the increment of T(m3-->2) of poly(dA).2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator-neomycin conjugates (1-4) to poly(dA).2poly(dT) was also confirmed by competition dialysis and a fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compounds 1-4 had slightly larger binding site size ( approximately 7-7.5) with poly(dA).2poly(dT) as compared to neomycin ( approximately 6.5). The thermodynamic parameters of these intercalator-neomycin conjugates with poly(dA).2poly(dT) were derived from an integrated van't Hoff equation using the T(m3-->2) values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA).2poly(dT) increased in the following order: neomycin < 1 < 3 < 4 < 2. Among them, the binding constant [(2.7 +/- 0.3) x 10(8) M(-1)] of 2 with poly(dA).2poly(dT) was the highest, almost 1000-fold greater than that of neomycin. The binding of compounds 1-4 with poly(dA).2poly(dT) was mostly enthalpy-driven and gave negative DeltaC(p) values. The results described here suggest that the binding affinity of intercalator-neomycin conjugates for poly(dA).2poly(dT) increases as a function of the surface area of the intercalator moiety.

Dataset Information

Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

Publications

Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

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