Design of DNA minor groove binding diamidines that recognize GC base pair sequences: a dimeric-hinge interaction motif

The classical model of DNA minor groove binding compounds is that they should have a crescent shape that closely fits the helical twist of the groove. Several compounds with relatively linear shape and large dihedral twist, however, have been found recently to bind strongly to the minor groove. These observations raise the question of how far the curvature requirement could be relaxed. As an initial step in experimental analysis of this question, a linear triphenyl diamidine, DB1111, and a series of nitrogen tricyclic analogues were prepared. The goal with the heterocycles is to design GC binding selectivity into heterocyclic compounds that can get into cells and exert biological effects. The compounds have a zero radius of curvature from amidine carbon to amidine carbon but a significant dihedral twist across the tricyclic and amidine-ring junctions. They would not be expected to bind well to the DNA minor groove by shape-matching criteria. Detailed DNase I footprinting studies of the sequence specificity of this set of diamidines indicated that a pyrimidine heterocyclic derivative, DB1242, binds specifically to a GC-rich sequence, -GCTCG-. It binds to the GC sequence more strongly than to the usual AT recognition sequences for curved minor groove agents. Other similar derivatives did not exhibit the GC specificity. Biosensor-surface plasmon resonance and isothermal titration calorimetry experiments indicate that DB1242 binds to the GC sequence as a highly cooperative stacked dimer. Circular dichroism results indicate that the compound binds in the minor groove. Molecular modeling studies support a minor groove complex and provide an inter-compound and compound-DNA hydrogen-bonding rational for the unusual GC binding specificity and the requirement for a pyrimidine heterocycle. This compound represents a new direction in the development of DNA sequence-specific agents, and it is the first non-polyamide, synthetic compound to specifically recognize a DNA sequence with a majority of GC base pairs.     

Minor groove binders and drugs targeting proteins cover complementary regions in chemical shape space

DNA minor groove binders (MGBs) are known to influence gene expression and are therefore widely studied to explore their therapeutic potential. We identified shape-based virtual screening with ROCS as a highly effective computational approach to enrich known MGBs in top-ranked molecules. Discovery of ten previously unknown MGBs by shape-based screening further confirmed the relevance of ligand shape for minor groove affinity. Based on experimental testing we propose three simple rules (at least two positive charges, four nitrogen atoms, and one aromatic ring) as filters to reach even better enrichment of true positives in ROCS hit lists. Interestingly, shape-based ranking of MGBs versus FDA-approved drugs again leads to high enrichment rates, indicating complementary coverage of chemical shape space and indicating minor groove affinity to be unfavorable for approval of drugs targeting proteins.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.     

DNA sequence dependent monomer-dimer binding modulation of asymmetric benzimidazole derivatives

A number of studies indicate that DNA sequences such as AATT and TTAA have significantly different physical and interaction properties. To probe these interaction differences in detail and determine the influence of charge, we have synthesized three bisbenzimidazole derivatives, a diamidine, DB185, and monoamidines, DB183 and DB210, that are related to the well-known minor groove agent, Hoechst 33258. Footprinting studies with several natural and designed DNA fragments indicate that the synthetic compounds bind at AT sequences in the minor groove and interact more weakly at sites with TpA steps relative to sites without such steps. Circular dichroism spectroscopy also indicates that the compounds bind in the DNA minor groove. Surprisingly, Tm studies as a function of ratio indicate that the monoamidines bind to TTAA sequences as dimers, whereas the diamidine binds as a monomer. Biosensor-surface plasmon resonance (SPR) studies allowed us to quantitate the interaction differences in more detail. SPR results clearly show that the monoamidine compounds bind to the TTAA sequence in a cooperative 2:1 complex but bind as monomers to AATT. The dication binds to both sequences in monomer complexes but the binding to AATT is significantly stronger than binding to TTAA. Molecular dynamics simulations indicate that the AATT sequence has a narrow time-average minor groove width that is a very good receptor site for the bisbenzimidazole compounds. The groove in TTAA sequences is wider and the width must be reduced to form a favorable monomer complex. The monocations thus form cooperative dimers that stack in an antiparallel orientation and closely fit the structure of the TTAA minor groove. The amidine groups in the dimer are oriented in the 5' direction of the strand to which they are closest. Charge repulsion in the dication apparently keeps it from forming the dimer. It instead reduces the TTAA groove width, in an induced fit process, sufficiently to form a minor groove complex. The dimer-binding mode of DB183 and DB210 is a new DNA recognition motif and offers novel design concepts for selective targeting of DNA sequences with a wider minor groove, including those with TpA steps.     

In silico footprinting of ligands binding to the minor groove of DNA

The sequence selectivity of small molecules binding to the minor groove of DNA can be predicted by "in silico footprinting". Any potential ligand can be docked in the minor groove and then moved along it using simple simulation techniques. By applying a simple scoring function to the trajectory after energy minimization, the preferred binding site can be identified. We show application to all known noncovalent binding modes, namely 1:1 ligand:DNA binding (including hairpin ligands) and 2:1 side-by-side binding, with various DNA base pair sequences and show excellent agreement with experimental results from X-ray crystallography, NMR, and gel-based footprinting.     

DNA binding studies of Vinca alkaloids: experimental and computational evidence

Fluorescence studies on the indole alkaloids vinblastine sulfate, vincristine sulfate, vincamine and catharanthine have demonstrated the DNA binding ability of these molecules. The binding mode of these molecules in the minor groove of DNA is non-specific. A new parameter of the purine-pyrimidine base sequence specificty was observed in order to define the non-specific DNA binding of ligands. Catharanthine had shown 'same' pattern of 'Pu-Py' specificity while evaluating its DNA binding profile. The proton resonances of a DNA decamer duplex were assigned. The models of the drug:DNA complexes were analyzed for DNA binding features. The effect of temperature on the DNA binding was also evaluated.     

DNA-templated dimerization of hairpin polyamides

Double-helical DNA accelerates the rate of ligation of two six-ring hairpin polyamides which bind adjacent sites in the minor groove via a 1,3-dipolar cycloaddition to form a tandem dimer. The rate of the templated reaction is dependent on DNA sequence as well as on the distance between the hairpin-binding sites. The tandem dimer product of the DNA-templated reaction has improved binding properties with respect to the smaller hairpin fragments. Since cell and nuclear uptake of DNA-binding polyamides will likely be dependent on size, this is a minimum first step toward the design of self-assembling small gene-regulating fragments to produce molecules of increasing complexity with more specific genomic targeting capabilities.     

Comparison of the binding stoichiometries of positively charged DNA-binding drugs using positive and negative ion electrospray ionization mass spectrometry

Positive and negative ion electrospray ionization (ESI) mass spectra of complexes of positively charged small molecules (distamycin, Hoechst 33258, [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2) have been compared. [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2 bind to DNA by intercalation. Negative ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA showed ions from DNA-ligand complexes consistent with solution studies. In contrast, only ions from free DNA were present in positive ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA, highlighting the need for obtaining ESI mass spectra of non-covalent complexes under a range of experimental conditions. Negative ion spectra of mixtures of the minor groove binder Hoechst 33258 with DNA containing a known minor groove binding sequence were dominated by ions from a 1:1 complex. In contrast, in positive ion spectra there were also ions present from a 2:1 (Hoechst 33258: DNA) complex, suggesting an alternative binding mode was possible either in solution or in the gas phase. When Hoechst 33258 was mixed with a DNA sequence lacking a high affinity minor groove binding site, the negative ion ESI mass spectra showed that 1:1 and 2:1 complexes were formed, consistent with existence of binding modes other than minor groove binding. The data presented suggest that comparison of positive and negative ion ESI-MS spectra might provide an insight into various binding modes in both solution and the gas phase.     

NMR structural analysis of a modular threading tetraintercalator bound to DNA

The synthesis and NMR structural studies are reported for a modular threading tetraintercalator bound to DNA. The tetraintercalator design is based on 1,4,5,8-tetracarboxylic naphthalene diimide units connected through flexible peptide linkers. Aided by an overall C(2) symmetry, NMR analysis verified a threading polyintercalation mode of binding, with linkers alternating in the order minor groove, major groove, minor groove, analogous to how a snake might climb a ladder. This study represents the first NMR analysis of a threading tetraintercalator and, as such, structurally characterizes a new topology for molecules that bind to relatively long DNA sequences with extensive access to both DNA grooves.     

Theoretical study of binding affinity for diamidine with DNA

Diamidine molecules, which have been recognized as the powerful gene drug candidates over the past decades, can bind in the DNA minor groove, inhibit the duplication of morbid sequences, and fight against a number of human and animal diseases. In this paper, on the basis of the binding models of a series of diamidines with DNA, the important influencing factors for the binding affinity of diamidines with DNA were systematically analyzed. The obtained results demonstrated that the curvature, length, distal group, and heteroaromatic ring of diamidine are four important factors, which could influence their binding affinities. Specifically, the better the curvature of the diamidine fits DNA minor groove, the higher the binding affinity is; increasing the molecular length within a certain range can make the binding affinity higher; changing the distal group of diamidine from amidino to imidazole or pyrimidine is favorable for improving the corresponding binding affinity; and the introduction of central heteroaromatic rings of diamidine molecules influences their binding affinities. One diamidine (named as DB103d) with ideal DNA binding affinity validates the four important factors proposed in the present work. The results obtained in this work might be helpful for the design of new efficient diamidine-based drug candidates.

     

Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study

The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.     

Fluorescence‐Labeled Bis‐benzamidines as Fluorogenic DNA Minor‐Groove Binders: Photophysics and Binding Dynamics

In recent decades there has been great interest in the design of highly sensitive sequence‐specific DNA binders. The eligibility of the binder depends on the magnitude of the fluorescence increase upon binding, related to its photophysics, and on its affinity and specificity, which is, in turn, determined by the dynamics of the binding process. Therefore, progress in the design of DNA binders requires both thorough photophysical studies and precise determination of the association and dissociation rate constants involved. We have studied two bis‐benzamidine (BBA) derivatives labeled by linkers of various lengths with the dye Oregon Green (OG). These fluorogenic binders show a dramatic fluorescence enhancement upon binding to the minor groove of double‐stranded (ds) DNA, as well as significant improvement in their sequence specificity versus the parent BBA, although with decreased affinity constants. Detailed photophysical analysis shows that static and dynamic quenching of the OG fluorescence by BBA through photoinduced electron transfer is suppressed upon insertion of BBA into the minor groove of DNA. Fluorescence correlation spectroscopy yields precise dynamic rate constants that prove that the association process of these fluorogenic binders to dsDNA is very similar to that of BBA alone and that their lower affinity is mainly a consequence of their weaker attachment to the minor groove and the resultant faster dissociation process. The conclusions of this study will allow us to go one step further in the design of new DNA binders with tunable fluorescence and binding properties.     

Short lexitropsin that recognizes the DNA minor groove at 5'-ACTAGT-3': understanding the role of isopropyl-thiazole

Isopropyl-thiazole ((iPr)Th) represents a new addition to the building blocks of nucleic acid minor groove-binding molecules. The DNA decamer duplex d(CGACTAGTCG)(2) is bound by a short lexitropsin of sequence formyl-PyPy(iPr)Th-Dp (where Py represents N-methyl pyrrole, (iPr)Th represents thiazole with an isopropyl group attached, and Dp represents dimethylaminopropyl). NMR data indicate ligand binding in the minor groove of DNA to the sequence 5'-ACT(5)AG(7)T-3' at a 2:1 ratio of ligand to DNA duplex. Ligand binding, assisted by the enhanced hydrophobicity of the (iPr)Th group, occurs in a head-to-tail fashion, the formyl headgroups being located toward the 5'-ends of the DNA sequence. Sequence reading is augmented through hydrogen bond formation between the exocyclic amine protons of G(7) and the (iPr)Th nitrogen, which lies on the minor groove floor. The B(I)/B(II) DNA backbone equilibrium is altered at the T(5) 3'-phosphate position to accommodate a B(II) configuration. The ligands bind in a staggered mode with respect to one another creating a six base pair DNA reading frame. The introduction of a new DNA sequence-reading element into the recognition jigsaw, combined with an extended reading frame for a small lexitropsin with enhanced hydrophobicity, holds great promise in the development of new, potentially commercially viable drug lead candidates for gene targeting.     

Major versus minor groove DNA binding of a bisarginylporphyrin hybrid molecule: A molecular mechanics investigation

On the basis of theoretical computations, we have recently synthesised [Perrée-Fauvet, M. and Gresh, N., Tetrahedron Lett., 36 (1995) 4227] a bisarginyl conjugate of a tricationic porphyrin (BAP), designed to target, in the major groove of DNA, the d(GGC GCC)2 sequence which is part of the primary binding site of the HIV-1 retrovirus site [Wain-Hobson, S. et al., Cell, 40 (1985) 9]. In the theoretical model, the chromophore intercalates at the central d(CpG)2 step and each of the arginyl arms targets O6/N7belonging to guanine bases flanking the intercalation site. Recent IR and UV-visible spectroscopic studies have confirmed the essential features of these theoretical predictions [Mohammadi, S. et al., Biochemistry, 37 (1998) 6165]. In the present study, we compare the energies of competing intercalation modes of BAP to several double-stranded oligonucleotides, according to whether one, two or three N- methylpyridinium rings project into the major groove. Correspondingly, three minor groove binding modes were considered, the arginyl arms now targeting N3, O2 sites belonging to the purine or pyrimidine bases flanking the intercalation site. This investigation has shown that: (i) in both the major and minor grooves, the best-bound complexes have the three N-methylpyridinium rings in the groove opposite to that of the phenyl group bearing the arginyl arms; (ii) major groove binding is preferred over minor groove binding by a significant energy (29 kcal/mol); and (iii) the best-bound sequence in the major groove is d(GGC GCC)2 with two successive guanines upstream from the intercalation. On the other hand, due to the flexibility of the arginyl arms, other GC-rich sequences have close binding energies, two of them being less stable than it by less than 8 kcal/mol. These results serve as the basis for the design of derivatives of BAP with enhanced sequence selectivities in the major groove.     

Use of Capillary Electrophoresis in the Study of Interaction between dsDNA and Drug Molecules

The advantages of berberine such as the anticancer1, antiinflammatory2 and no side effects of camptothecin1, have promoted the research in the mechanism of berberine with macrobiomolecules. In general, three different points of view have been presented on…     

Structural and dynamic variations in DNA hexamers containing T-T and F-F single and tandem internal mispairs

Molecular dynamics simulations of double-helical DNA oligomers have been performed to investigate differences in the structure, dynamics, and hydration of F-F and T-T mispairs. Hexamers containing F-F pairs were found to be more dynamic, especially in the region of the mispair itself. This dynamic variability derives from greater flexibility of F-F pairs. The T-T mispairs, on the other hand, were found to be comparatively tightly bound as wobble pairs. The major and minor groove edges of the T-T pairs were observed to be solvated at exposed carbonyl positions by at least one water molecule, while F-F pairs lacked solvating waters. Stacking interactions were nearly identical for T-T and F-F pairs, leading to similar average structures, even though F stacking was more dynamically variable. Solvation differences between F-F and T-T therefore support the steric exclusion model for nucleotide incorporation in DNA replication. Large differences in the orientation of minor groove functional groups, in addition to differences in solvation, further rationalize why F bases present during DNA extension events induce stalls. Two novel nucleotides are proposed to further elucidate minor groove interactions of DNA with polymerase molecules.Electronic Supplementary Material This Material consists of equilibration protocol, plots of center-of-mass stacking, water radial distribution functions, helical parameter dynamics, and dynamics data for a control AT sequence. Supplementary material is available in the online version of this article at Contribution to the Jacopo Tomasi Honorary Issue