Cu2+ -mediated assembly of the minor groove binders on the DNA template with sequence selectivity

The ligands (Bpy-H, 5) have been designed to connect the Hoechst33258 skeleton for DNA binding and 2,2'-bipyridine for Cu(2+) complexation. It has been revealed that the new Hoechst ligand long Bpy-H (5L) having a long linker exhibits Cu(2+)-mediated assembly on the DNA template having two A(3)T(3) sites in a selective manner depending on the length of the linker of the ligand as well as on the distance between the two A(3)T(3) sites of DNA. [reaction: see text]     

Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting amyloid beta formation: in silico and in vitro study

Combining Lipinski’s rule with the docking and steered molecular dynamics simulations and using the PubChem data base of about 1.4 million compounds, we have obtained DNA dyes Hoechst 34580 and Hoechst 33342 as top-leads for the Alzheimer’s disease. The binding properties of these ligands to amyloid beta (Aβ) fibril were thoroughly studied by in silico and in vitro experiments. Hoechst 34580 and Hoechst 33342 prefer to locate near hydrophobic regions with binding affinity mainly governed by the van der Waals interaction. By the Thioflavin T assay, it was found that the inhibition constant IC50 ≈ 0.86 and 0.68 μM for Hoechst 34580 and Hoechst 33342, respectively. This result qualitatively agrees with the binding free energy estimated using the molecular mechanic-Poisson Boltzmann surface area method and all-atom simulations with the AMBER-f99SB-ILDN force field and water model TIP3P. In addition, DNA dyes have the high capability to cross the blood brain barrier. Thus, both in silico and in vitro experiments have shown that Hoechst 34580 and 33342 are good candidates for treating the Alzheimer’s disease by inhibiting Aβ formation.     

Interaction with plasmid DNA of Hoechst-TACN conjugates

ABSTRACT

A new family of conjugates between the Hoechst minor groove binder and the TACN metal ion ligand connected through hydrophobic alkyl or more hydrophilic oxyethyl linkers of different length has been prepared. The linkers are connected to the convex side of the Hoechst skeleton thus forcing the TACN ligand to exit the minor groove and interact with the phosphate backbone of DNA. The conjugates preserve the binding mode of Hoechst with an affinity influenced by the nature of the linker, the more hydrophobic being the more efficient. Coordination of Cu(II) or Zn(II) poorly affect these parameters. Nevertheless, the Zn(II) complex bearing a C6 linear alkyl linker induced a modest but reproducible acceleration of the hydrolytic cleavage of DNA which can be ascribed to the ability of the conjugate to deliver the hydrolytic subunit close to the DNA phosphodiester bonds.     

SAR by MS

RNAs have recently emerged as an exciting new target for small molecule therapeutics. Conventional HTS discovery strategies measuring disruption of RNAprotein interactions have proven unsuccessful. We describe a ligand-based drug discovery strategy that addresses the inherent difficulties RNA targets. The strategy is based on: 1) using a MS spectrometry (MS)-based assay to measure the affinity of compounds for a target; 2) performing competitive binding experiments and molecular modeling with the motifs to determine the binding site(s) of the ligands; 3) design and synthesis of derivatives of interesting binders to establish the linking sites; 4) identifying the appropriate linker group using MS; 5) fusing motifs into a more complex structure to afford higher affinity compounds. Example of applying this strategy to identify new classes of lead molecules with affinity and specificity for ribosomal RNA targets will be presented.     

Multiple binding modes for dicationic Hoechst 33258 to DNA

The binding of dicationic Hoechst 33258 (ligand) to DNA was characterized by means of the fluorescence spectra, fluorescence intensity titration, time-resolved fluorescence decay, light scattering, circular dichroism, and fluorescence thermal denaturation measurements, and two binding modes were distinguished by the experimental results. Type 1 binding has the stoichiometry of one ligand to more than 12 base pairs, and it is defined as quasi-minor groove binding which has the typical prolonged fluorescence lifetime of about 4.4 ns. In type 1 binding, planar conformation of the ligand is favorable. Type 2 binding with phosphate to ligand ratio (P/L) < 2.5 has the stoichiometry of one ligand to two phosphates. It is defined as a highly dense and orderly stacked binding with DNA backbone as the template. Electrostatic interactions between doubly protonated ligands and negatively charged DNA backbone play a predominant role in the type 2 binding mode. The characteristics of this type of binding result in a twisted conformation of the ligand that has a fluorescence lifetime of less than 1 ns. The results also indicate that the binding is in a cooperative manner primarily by stacking of the aromatic rings of the neighboring ligands. Type 1 binding is only observed for double-stranded DNA (dsDNA) with affinity constant of 1.83 x 10(7) M-1. In the type 2 binding mode, the binding affinity constants are 4.9 x 10(6) and 4.3 x 10(6) M-1 for dsDNA and single-stranded DNA (ssDNA), respectively. The type 2 binding is base pair independent while the type 1 binding is base pair related. The experiments described in this paper revealed that the dication bindings are different from the monocation bindings reported by previous study. The dication binding leads to stronger aggregation at low ligand concentration and results in orderly arrangements of the ligands along DNA chains. Furthermore the dication binding is demonstrated to be beneficial for enhancing the DNA's stability.     

Affinity NMR: decoding DNA binding

We have shown that affinity NMR can be used to edit a NMR spectrum so that ligands that have affinity to DNA can be observed in the presence of other nonbinding molecules. Diffusion encoded spectroscopy (DECODES) can be used to identify the binding ligands. We were able to identify Hoechst 33342 as binding to the Drew-Dickerson dodecamer d(CGCGAATTCGCG)2 in the presence of the nonbinding molecules adenine, adenosine, and thiamine. Affinity NMR appears to be readily applicable to DNA systems for the following reasons. (1) The relaxation rate of the DNA oligonucleotides is favorable, thus the signal intensity loss due to relaxation is not severe. (2) A comparison of the patterns of the DNA cross-peaks upon binding in the two-dimensional total correlation spectroscopy and correlation spectroscopy spectrum are easily performed, and the ligand signals in the two-dimensional DECODES spectrum can be readily identified. (3) The aromatic part of the DNA spectrum is devoid of 2D cross-peaks in these correlation spectra, greatly facilitating the interpretation of the bound ligand in the DECODES spectrum.     

Design, synthesis, and validation of a branched flexible linker for bioactive peptides

A branched flexible linker that incorporates a fluorescent dansyl moiety was synthesized and used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The linker was incorporated into the conjugate by solid-phase synthesis. In vitro biological evaluations showed that potency of binding to the human melanocortin 4 receptor was not diminished for linker-ligand combinations relative to the corresponding ligand alone.     

Extending the language of DNA molecular recognition by polyamides: unexpected influence of imidazole and pyrrole arrangement on binding affinity and specificity

Pyrrole (Py) and imidazole (Im) polyamides can be designed to target specific DNA sequences. The effect that the pyrrole and imidazole arrangement, plus DNA sequence, have on sequence specificity and binding affinity has been investigated using DNA melting (DeltaT(M)), circular dichroism (CD), and surface plasmon resonance (SPR) studies. SPR results obtained from a complete set of triheterocyclic polyamides show a dramatic difference in the affinity of f-ImPyIm for its cognate DNA (K(eq) = 1.9 x 10(8) M(-1)) and f-PyPyIm for its cognate DNA (K(eq) = 5.9 x 10(5) M(-1)), which could not have been anticipated prior to characterization of these compounds. Moreover, f-ImPyIm has a 10-fold greater affinity for CGCG than distamycin A has for its cognate, AATT. To understand this difference, the triamide dimers are divided into two structural groupings: central and terminal pairings. The four possible central pairings show decreasing selectivity and affinity for their respective cognate sequences: -ImPy > -PyPy- > -PyIm- approximately -ImIm-. These results extend the language of current design motifs for polyamide sequence recognition to include the use of "words" for recognizing two adjacent base pairs, rather than "letters" for binding to single base pairs. Thus, polyamides designed to target Watson-Crick base pairs should utilize the strength of -ImPy- and -PyPy- central pairings. The f/Im and f/Py terminal groups yielded no advantage for their respective C/G or T/A base pairs. The exception is with the -ImPy- central pairing, for which f/Im has a 10-fold greater affinity for C/G than f/Py has for T/A.     

Determination of Allosteric Effects and Interstrand Bidentate Interactions in DNA‐Peptide Molecular Recognition

To study DNA allostery, quantitative DNase I footprinting studies were carried out on a newly designed peptide His‐Hyp‐Lys‐Lys‐(Py)₄‐Lys‐Lys‐NH₂ (HypKK‐10) containing the XHypKK (Hyp = hydroxyproline) and polyamide motifs. The interconnection of DNA footprints of peptides HypKK‐10 and the parent peptide PyPro‐12 supports the proposal that interaction network cooperativity is preferred in DNA‐peptide interactions between multiple recognition sites. A simple method of determining interstrand bidentate interactions between the peptide moieties and DNA bases is introduced. It is envisaged that interstrand bidentate interactions also participate in the relay of conformational changes to recognition sites on the complementary strands. Circular dichroism studies of the titration of peptide HypKK‐10 with an oligonucleotide duplex indicate that this peptide binds in a dimeric fashion to DNA in the minor groove. This work may prompt the design of new DNA binding ligands for the study of DNA‐peptide allosteric interactions and DNA interaction network.     

Influence of terminal substitution on structural, DNA, protein binding, anticancer and antibacterial activities of palladium(II) complexes containing 3-methoxy salicylaldehyde-4(N) substituted thiosemicarbazones

The variable chelating behavior of 3-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones was observed in equimolar reactions with [PdCl(2)(PPh(3))(2)]. The new complexes were characterized by various analytical, spectroscopic techniques (mass, (1)H-NMR, absorption, IR). All the new complexes were structurally characterized by single crystal X-ray diffraction. Crystallographic results showed that the ligands H(2)L(1) and H(2)L(4) are coordinated as binegative tridentate ONS donor ligands in the complexes 1 and 4 by forming six and five member rings. However, the ligands H(2)L(2) and H(2)L(3) bound to palladium in 2 and 3 as uninegative bidentate NS donors by forming a five member chelate ring. From this study, it was found that the substitution on terminal 4(N)-nitrogen may have an influence on the chelating ability of thiosemicarbazone. The presence of hydrogen bonding in 2 and 3 might be responsible for preventing the coordination of phenolic oxygen to the metal ion. The interaction of the complexes with calf-thymus DNA (CT-DNA) has been explored by absorption and emission titration methods. Based on the observations, an electrostatic binding mode of DNA has been proposed. The protein binding studies were monitored by quenching of tryptophan and tyrosine residues in the presence of complexes using Lysozyme as model protein. Antibacterial activity studies of the complexes have been screened against pathogenic bacteria such as Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. MIC50 values of the complexes showed that they exhibited significant activity against the pathogens and among them, 3 exhibited higher activity. Further, anticancer activity of the complexes on the lung cancer cell line A549 has also been studied.     

Synthesis, characterization, and crystal structures of three new divalent metal carboxylate-sulfonates with a layered and one-dimensional structure

Hydrothermal reactions of 5-sulfoisophthalic acid (HO(3)SC(6)H(3)-1,3-(CO(2)H)(2), H(3)L) with M(II) carbonate (or oxide) and 4,4'-bipyridine (4,4'-bipy) (or 2,2'-bipyridine, 2,2'-bipy) resulted in three new metal carboxylate-sulfonate hybrids, namely, [CdL(H-4,4'-bipy)] (1) and [Cd(3)L(2)(2,2-bipy)(4)(H(2)O)(2)].2H(2)O (2) with layered structures and [ZnL(H-4,4'-bipy)(H(2)O)].2H(2)O (3), whose structure features a one-dimensional double chain. The cadmium(II) ion in complex 1 is seven-coordinated by five carboxylate oxygen atoms and one sulfonate oxygen atom from four ligands and a unidentate 4,4'-bipyridine. The interconnection of the cadmium(II) ions through bridging carboxylate-sulfonate ligands resulted in the formation of a <002> double layer with the bipyridyl rings orientated toward the interlayer space. Complex 2 has a different layered structure. Cd(1) is seven-coordinated by two bidentate chelating carboxylate groups from two ligands, a bidentate chelating 2,2'-bipy and an aqua ligand, and Cd(2) is octahedrally coordinated by two bidentate chelating 2,2'-bipy's, a sulfonate oxygen, and an aqua ligand. The coordination geometry around Cd(3) is similar to that of Cd(1) with the aqua ligand being replaced by an oxygen atom from the sulfonate group. The carboxylate-sulfonate ligand acts as pentadentate ligand, bridging with three cadmium(II) ions. The bridging of cadmium(II) ions through the carboxylate-sulfonate ligands resulted in the formation of <006> and <003> layers; the 2,2'-bipy molecules and [Cd(2)(2,2'-bipy)(2)(H(2)O)] cations are orientated to the interlayer space. Complex 3 features a 1D metal carboxylate-sulfonate double chain along the diagonal of the a- and b-axes. The zinc(II) ion is octahedrally coordinated by four carboxylate O atoms from three ligands, a unidentate 4,4'-bipy, and an aqua ligand. Each pair of zinc(II) ions is bridged by two carboxylate groups from two ligands to form a dimer, and such dimeric units are interconnected by bridging ligands to form a double chain. The sulfonate group of the carboxylate sulfonate ligand remains noncoordinated and forms a number of hydrogen bonds with aqua ligands as well as lattice water molecules.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

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…     

Parallel synthesis of H-pin polyamides by alkene metathesis on solid phase

A small library of H-pin polyamides with variable aliphatic bridge lengths (CH(2))(n)(), where n = 4-8, connecting a central Py/Py pair was prepared via parallel synthesis with Ru-catalyzed alkene metathesis on solid phase as a complexity-generating cross-linking reaction. DNA binding affinities and sequence specificities were analyzed for each member of the library to determine the optimum linker length. An H-pin polyamide with a six-methylene bridge was found to have the highest affinity to its match site with high selectivity over a 1-bp mismatch site. The relationship between the number of methylenes in the linker (CH(2))(n)() and affinity is n = 6 > 4 > 7 > 5 > 8. These results indicate that 6 followed by 4 methylene-bridged polyamides represent the optimum spacer length for the H-pin motif in the DNA minor groove. Importantly, the H-pin is competitive with hairpin polyamides with respect to affinity and specificity. The metathesis-based convergent synthetic route to H-pin polyamides expands the scope of readily available DNA recognition motifs for small molecule-based gene regulation studies.     

Synthesis and spectroelectrochemistry of Ir(bpy)(phen)(phi)(3+), a tris(heteroleptic) metallointercalator

A tris(heteroleptic) phenanthrenequinone diimine (phi) complex of Ir(III), Ir(bpy)(phen)(phi)(3+), was synthesized through the stepwise introduction of three different bidentate ligands, and the Lambda- and Delta-enantiomers were resolved and characterized by CD spectroscopy. Like other phi complexes, this tris(heteroleptic) iridium complex binds avidly to DNA by intercalation. Electrochemical studies show that Ir(bpy)(phen)(phi)(3+) undergoes a reversible one-electron reduction at E(0) = -0.025 V in 0.1 M TBAH/DMF (versus Ag/AgCl), and spectroelectrochemical studies indicate that this reduction is centered on the phi ligand. The EPR spectrum of electrochemically generated Ir(bpy)(phen)(phi)(2+) is consistent with a phi-based radical. The electrochemistry of Ir(bpy)(phen)(phi)(3+) was also probed at a DNA-modified electrode, where a DNA binding affinity of K = 1.1 x 10(6) M(-1) was measured. In contrast to Ir(bpy)(phen)(phi)(3+) free in solution, the complex bound to DNA undergoes a concerted two-electron reduction, to form a diradical species. On the basis of UV-visible and EPR spectroscopies, it is found that disproportionation of electrochemically generated Ir(bpy)(phen)(phi)(2+) occurs upon DNA binding. These results underscore the rich redox chemistry associated with metallointercalators bound to DNA.     

Osmolyte changes the binding affinity and mode of interaction of minor groove binder hoechst 33258 with calf thymus DNA

The aim of this work was to investigate the effect of altered water activity on Hoechst 33258-calf thymus DNA (CtDNA) interaction by using osmotic stress approach. Water activity was changed by using osmolytes viz., sucrose and triethylene glycol (TEG). We have reported the results of thermal denaturation, absorption and fluorescence spectroscopy and binding affinity measurements as a function of osmolytes concentration. TEG dramatically lowered the thermal stability of CtDNA, ΔT(m)=-16 °C whereas sucrose induced very little decrease. Hoechst 33258 increases the stability of CtDNA, but in the presence of TEG, the ΔT(m) was -37 °C and a marginal decrease was observed with sucrose. Binding affinity of Hoechst 33258 with CtDNA was found to be reduced from 4.75×10? to 0.16×10? M?1 in TEG and this was accompanied with the increased uptake of 74±2 water molecules. In the presence of sucrose this uptake of water molecules was found to be 30±1. Method of continuous variation suggests that the osmolytes lowered the stoichiometry of Hoechst 33258-CtDNA complex. On the contrary, van't Hoff plot revealed the hydrophobic interaction (ΔS=130.66 J mol?1 K?1) between the Hoechst 33258 and CtDNA. The detailed absorption and fluorescence spectral measurements including the fluorescence lifetime and anisotropy indicated bound state of Hoechst 33258 in osmotic stress condition. Fluorescence lifetime measurement revealed that the contribution from the planar conformer of Hoechst 33258 dominated the binding interaction with CtDNA in presence of TEG. These results can be useful for understanding of interaction of Hoechst 33258 with genomic DNA in a complex environment having altered water activity.     

Coordination driven self-assembly of four new molecular boats using a flexible imidazole-containing donor linker

Using a metal-ligand coordination bonding approach, the self-assembly of four new metallamacrocycles from Pd(ii)-based 90 degrees acceptors and a diimidazole donor ligand 1,3-bis(imidazole-1-ylmethyl)-2,4,6-trimethylbenzene (L) has been achieved. The assemblies are characterized fully by NMR and electrospray ionization-mass spectroscopic (ESI-MS) analysis and in two cases the X-ray single-crystal structure analysis established the gross structures. The selective formation of a diimidazole-based linker (L) containing macrocycle [(en)Pd(micro-L)2Pd(en)]4+ from a 1 : 1 : 1 mixture of cis-Pd(en)(NO3)2, and 1,2-bis(4-pyridyl)ethane is also established. Measuring the binding constants established the stronger Pd- binding force compared to traditional Pd-N(pyridyl linker) interaction, which reveals the possibility of using imidazole donor ligands as potential linkers or even better ligands compared to the widely used pyridyl donor ligands in the construction of metal-based large supramolecular assemblies.     

Multivalency effects in protein--carbohydrate interaction: the binding of the Shiga-like toxin 1 binding subunit to multivalent C-linked glycopeptides

A series of monovalent and bivalent glycopeptides displaying a C-linked analogue of the Pk trisaccharide, the in vivo ligand for the pentavalent Shiga-like toxin binding subunit (SLT-1B), were prepared and evaluated as ligands for SLT-1B by isothermal titration microcalorimetry and competitive enzyme-linked immunosorbent assay (ELISA). Although none of the monovalent ligands showed any enhancement in affinity compared to O-methyl glycoside, two bivalent ligands show significant enhancements in affinity in assays. This observation represents the first calorimetric observation of an enhancement in affinity for this system. In contrast, only one of the two ligands shows an enhancement in the competitive ELISA. Together, these data signal a difference in the means by which the two ligands achieve affinity, apparently triggered by a change in the nature of the linker domain. These results provide a rationalization for apparently contradictory reports from the recent literature and again emphasize the importance of investigating complex binding phenomena by multiple techniques.     

Application of NMR screening techniques for observing ligand binding with a protein receptor

Water ligand observed via gradient spectroscopy (WaterLOGSY), saturation transfer difference and NOE pumping NMR techniques were used to identify ligand binding with a receptor. Although these experiments were originally designed to observe ligands in complexes, their application is limited by the affinity of ligands towards target molecules. Here the improved WaterLOGSY pulse sequence was developed by incorporating the double pulsed field gradient spin-echo and gradient-tailored excitation WATERGATE sequences. The efficiency of these ligand-observed NMR screening techniques was investigated using the ribonuclease T1-inhibitor system.     

Synthetic dsDNA‐Binding Peptides Using Natural Compounds as Model

We have developed a series of short DNA‐binding peptides containing newly synthesized, unnatural as well as natural amino acid building blocks. By a combinatorial‐library approach, oligopeptides were developed with moderate dsDNA‐binding affinities. Two strategies were used to further enhance the binding affinity of the lead peptides: Ac‐Arg‐Ual‐Sar‐Chi‐Chi‐Chi‐Arg‐NH₂ and Ac‐Arg‐Cbg‐Cha‐Chi‐Chi‐Tal‐Arg‐NH₂. Site‐selective amino acid substitutions increased the binding affinities up to 2 × 10^?5 M . Further enhancement of the binding affinities could be achieved by coupling of an acridine intercalating unit, using linker arms of different length and flexibility. With the introduction of a new lysine‐based acridine unit, different types of oligopeptide–acridine conjugates were designed using known dsDNA‐binding ligands as model compounds. The binding capacities of these new oligopeptide–acridine conjugates have been investigated by a fluorescent intercalator (ethidium bromide) displacement (FID) assay. With the synthesis of the dipeptide–acridine conjugates, binding affinities in the low micromolar range were obtained (6.4 × 10^?6 M ), which is similar to the binding strength of the well‐known DNA binder Hoechst 33258.