A novel approach using electrospray ionization mass spectrometry to study competitive binding of small molecules with mixed DNA sequences.
ABSTRACT: Minor groove binding compounds have been shown to induce changes in global DNA conformation, allosterically inhibiting DNA-protein interactions necessary for transcriptional processes. Many minor groove binders are specific for AT base pairs but have little preference over alternating AT or A-tract sequences. Few compounds, other than polyamides, show selectivity for mixed sequences with AT and GC base pairs. Electrospray ionization mass spectrometry (ESI-MS) can provide insight on the stoichiometry and relative affinities in minor groove recognition of different DNA sequences with a library of minor groove binders. A goal in our current research is to develop new compounds that recognize mixed sequences of DNA. In an effort to optimize screening for compounds that target mixed AT and GC base pair sequences of DNA, ESI-MS was used to study the competitive binding of compounds with a mixed set of DNA sequences. The method identified preferred binding sites, relative affinities, and concentration-dependent binding stoichiometry for the minor groove binding compounds netropsin and DB75 with AT-rich sequences and DB293 with ATGA and AT sites.
Project description:Small-molecule targeting of the DNA minor groove is a promising approach to modulate genomic processes necessary for normal cellular function. For instance, dicationic diamindines, a well-known class of minor groove binding compounds, have been shown to inhibit interactions of transcription factors binding to genomic DNA. The applications of these compounds could be significantly expanded if we understand sequence-specific recognition of DNA better and could use the information to design more sequence-specific compounds. Aside from polyamides, minor groove binders typically recognize DNA at A-tract or alternating AT base pair sites. Targeting sites with GC base pairs, referred to here as mixed base pair sequences, is much more difficult than those rich in AT base pairs. Compound 1 is the first dicationic diamidine reported to recognize a mixed base pair site. It binds in the minor groove of ATGA sequences as a dimer with positive cooperativity. Due to the well-characterized behavior of 1 with ATGA and AT rich sequences, it provides a paradigm for understanding the elements that are key for recognition of mixed sequence sites. Electrospray ionization mass spectrometry (ESI-MS) is a powerful method to screen DNA complexes formed by analogues of 1 for specific recognition. We also report a novel approach to determine patterns of recognition by 1 for cognate ATGA and ATGA-mutant sequences. We found that functional group modifications and mutating the DNA target site significantly affect binding and stacking, respectively. Both compound conformation and DNA sequence directionality are crucial for recognition.
Project description:Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, have found extensive uses in biotechnology, and are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds have shown us that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, and the compounds can form cooperative stacked dimers to recognize GC and mixed AT/GC base pair sequences.
Project description:AT specific heterocyclic cations that bind in the DNA duplex minor groove have had major successes as cell and nuclear stains and as therapeutic agents which can effectively enter human cells. Expanding the DNA sequence recognition capability of the minor groove compounds could also expand their therapeutic targets and have an impact in many areas, such as modulation of transcription factor biological activity. Success in the design of mixed sequence binding compounds has been achieved with N-methylbenzimidazole ( N-MeBI) thiophenes which are preorganized to fit the shape of the DNA minor groove and H-bond to the -NH of G·C base pairs that project into the minor groove. Initial compounds bind strongly to a single G·C base pair in an AT context with a specificity ratio of 50 ( KD AT-GC/ KD AT) or less and this is somewhat low for biological use. We felt that modifications of compound shape could be used to probe local DNA microstructure in target mixed base pair sequences of DNA and potentially improve the compound binding selectivity. Modifications were made by increasing the size of the benzimidazole N-substituent, for example, by using N-isobutyl instead of N-Me, and by changing the molecular twist by introducing substitutions at specific positions on the aromatic core of the compounds. In both cases, we have been able to achieve a dramatic increase in binding specificity, including no detectible binding to pure AT sequences, without a significant loss in affinity to mixed base pair target sequences.
Project description:A series of symmetrical dimeric proton pump inhibitor (PPI) analogues, designed as novel type DNA minor groove binders, was synthesized and evaluated for anti-tumor activity. Some of these new compounds showed IC(50) values below 10 microM in an in vitro anti-tumor test. A molecular modeling study was performed to confirm the sequence selectivity of these compounds towards AT base pairs in DNA. Two effective compounds were selected and docked into the minor groove of DNA. The snug binding may be responsible for their cytotoxic and anti-tumor effects.
Project description:The compounds synthesized in this research were designed with the goal of establishing a new paradigm for mixed-base-pair DNA sequence-specific recognition. The design scheme starts with a cell-permeable heterocyclic cation that binds to AT base pair sites in the DNA minor groove. Modifications were introduced in the original compound to include an H-bond accepting group to specifically recognize the G-NH that projects into the minor groove. Therefore, a series of heterocyclic cations substituted with an azabenzimidazole ring has been designed and synthesized for mixed-base-pair DNA recognition. The most successful compound, 12a, had an azabenzimidazole to recognize G and additional modifications for general minor groove interactions. It binds to the DNA site -AAAGTTT- more strongly than the -AAATTT- site without GC and indicates the design success. Structural modifications of 12a generally weakened binding. The interactions of the new compound with a variety of DNA sequences with and without GC base pairs were evaluated by thermal melting analysis, circular dichroism, fluorescence emission spectroscopy, surface plasmon resonance, and molecular modeling.
Project description:The structural and thermodynamic basis for the strength and selectivity of the interactions of minor groove binders (MGBs) with DNA is not fully understood. In 2003, we reported the first example of a thiazole-containing MGB that bound in a phase-shifted pattern that spanned six base pairs rather than the usual four (for tricyclic distamycin-like compounds). Since then, using DNA footprinting, NMR spectroscopy, isothermal titration calorimetry, and molecular dynamics, we have established that the flanking bases around the central four being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences.
Project description:Biophysical studies have been carried out on a family of asymmetric guanidinium-based diaromatic derivatives to assess their potential as DNA minor groove binding agents. To experimentally assess the binding of these compounds to DNA, solution phase biophysical studies have been performed. Thus, surface plasmon resonance, UV-visible spectroscopy and circular and linear dichroism have been utilized to evaluate binding constants, stoichiometry and mode of binding. In addition, the thermodynamics of the binding process have been determined by using isothermal titration calorimetry. These results show significant DNA binding affinity that correlates with the expected 1?:?1 binding ratio usually observed for minor groove binders. Moreover, a simple computational approach has been devised to assess the potential as DNA binders of this family of compounds.
Project description:Heterocyclic diamidines are strong DNA minor-groove binders and have excellent antiparasitic activity. To extend the biological activity of these compounds, a series of arylimidamides (AIAs) analogues, which have better uptake properties in Leishmania and Trypanosoma cruizi than diamidines, was prepared. The binding of the AIAs to DNA was investigated by Tm , fluorescence displacement titration, circular dichroism, DNase I footprinting, biosensor surface plasmon resonance, X-ray crystallography and molecular modeling. These compounds form 1:1 complexes with AT sequences in the DNA minor groove, and the binding strength varies with substituent size, charge and polarity. These substituent-dependent structure and properties provide a SAR that can be used to estimate K values for binding to DNA in this series. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for AIAs.
Project description:Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. The heterocyclic diamidine, DB2277, specifically recognizes a single G•C base pair in the minor groove of mixed base pair sequences of the type AAAGTTT. NMR spectroscopy reveals the presence of major and minor species of the bound compound. To understand the principles that determine the binding affinity and orientation in mixed sequences of DNA, over thirty DNA hairpin substrates were examined by NMR and thermal melting. The NMR exchange dynamics between major and minor species shows that the exchange is much faster than compound dissociation determined from biosensor-surface plasmon resonance. Extensive modifications of DNA sequences resulted in a unique DNA sequence with binding site AAGATA that binds DB2277 in a single orientation. A molecular docking result agrees with the model representing rapid flipping of DB2277 between major and minor species. Imino spectral analysis of a (15)N-labeled central G clearly shows the crucial role of the exocyclic amino group of G in sequence-specific recognition. Our results suggest that this approach can be expanded to additional modules for recognition of more sequence-specific DNA complexes. This approach provides substantial information about the sequence-specific, highly efficient, dynamic nature of minor groove binding agents.
Project description:DNA is the target of chemical compounds (drugs, pollutants, photosensitizers, etc.), which bind through non-covalent interactions. Depending on their structure and their chemical properties, DNA binders can associate to the minor or to the major groove of double-stranded DNA. They can also intercalate between two adjacent base pairs, or even replace one or two base pairs within the DNA double helix. The subsequent biological effects are strongly dependent on the architecture of the binding motif. Discriminating between the different binding patterns is of paramount importance to predict and rationalize the effect of a given compound on DNA. The structural characterization of DNA complexes remains, however, cumbersome at the experimental level. In this contribution, we employed all-atom molecular dynamics simulations to determine the standard binding free energy of DNA with netropsin, a well-characterized antiviral and antimicrobial drug, which associates to the minor groove of double-stranded DNA. To overcome the sampling limitations of classical molecular dynamics simulations, which cannot capture the large change in configurational entropy that accompanies binding, we resort to a series of potentials of mean force calculations involving a set of geometrical restraints acting on collective variables.