Dimeric and trimeric derivatives of the azinomycin B chromophore show enhanced DNA binding.
ABSTRACT: To explore the utility of the azinomycin B chromophore as a platform for the development of major-groove binding small molecules, we have prepared a series of 3-methoxy-5-methylnaphthalene derivatives containing diamine, triamine, and carbohydrate linker moieties. All bis- and tris-azinomycin derivatives are intercalators that display submicromolar binding affinities for calf-thymus DNA, as revealed by viscometry measurements and fluorescent intercalator displacement (FID) assays, respectively. Although the tightest binding ligand 1d (Ka = 2.42 × 107 M-1) has similar affinities for sequence diverse polynucleotides, competition binding studies with methylated phage DNA and known major and minor groove binding small molecules suggest that the tether moiety linking the naphthalene chromophores may occupy the major groove of DNA.
Project description:Monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a-1f) have been synthesized for the purpose of evaluating their affinity and sequence selectivity for duplex DNA. Competitive ethidum displacement assays indicate that 1a-1f have apparent association constants for CT DNA in the range of 1.80-16.2?×?107?M-1 and binding site sizes of 10-14?bp. Viscosity experiments performed on ligand 1f confirmed that these dyes associate with duplex DNA by a non-intercalative mode of binding. Circular dichroism and competition binding studies of the tightest binding ligand 1e with known major and minor groove binding molecules suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers, suggesting a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove.
Project description:Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine-pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.
Project description:A series of N-monosubstituted and N,N'-disubstituted derivatives of the indolo[3,2-b]carbazole chromophore have been prepared, and their binding affinity for duplex DNA has been evaluated by ultraviolet and fluorescence spectroscopies. It has been found that indolo[3,2-b]carbazoles bearing basic N-alkyl substituents are intercalators that bind DNA with affinities in the micromolar and submicromolar range and a preference for associating with sequences of mixed composition and purine-pyrimidine steps.
Project description:XR5944 is a potent anticancer drug with a novel DNA binding mode: DNA bisintercalationg with major groove binding. XR5944 can bind the estrogen response element (ERE) sequence to block ER-ERE binding and inhibit ER? activities, which may be useful for overcoming drug resistance to currently available antiestrogen treatments. This review discusses the progress relating to the structure and function studies of specific DNA recognition of XR5944. The sites of intercalation within a native promoter sequence appear to be different from the ideal binding site and are context- and sequence- dependent. The structural information may provide insights for rational design of improved EREspecific XR5944 derivatives, as well as of DNA bis-intercalators in general.
Project description:Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein-DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.
Project description:The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities.
Project description:To better understand the molecular basis for recognition of the DNA minor groove by heterocyclic cations, a series of "reversed amidine" substituted heterocycles has been prepared. Amidine derivatives for targeting the minor groove have the amidine carbon linked to a central heterocyclic system, whereas in the reverse orientation, an amidine nitrogen provides the link. The reverse system has a larger dihedral angle as well as a modified spatial relationship with the groove relative to amidines. Because of the large dihedral, the reversed amidines should have reduced binding to DNA relative to similar amidines. Such a reduction is observed in footprinting, circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and isothermal titration calorimetric (ITC) experiments with DB613, which has a central phenyl-furan-phenyl heterocyclic system. The reduction is not seen when a pyrrole (DB884) is substituted for the furan. Analysis of a number of derivatives defines the pyrrole and a terminal phenyl substituent on the reversed amidine groups as critical components in the strong binding of DB884. ITC and SPR comparisons showed that the better binding of DB884 was due to a more favorable binding enthalpy and that it had exceptionally slow dissociation from DNA. Crystallographic analysis of DB884 bound to an AATT site shows that the compound was bound in the minor groove in a 1:1 complex as suggested by CD solution studies. Surprisingly, unlike the amidine derivative, the pyrrole -NH of DB884 formed an H-bond with a central T of the AATT site and this accounts for the enthalpy-driven strong binding. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for related amidine and reversed amidine analogues.
Project description:The use of di(2-pyridyl)ketone in subcomponent self-assembly is introduced. When combined with a flexible triamine and zinc bis(trifluoromethanesulfonyl)imide, this ketone formed a new Zn4 L4 tetrahedron 1 bearing twelve uncoordinated pyridyl units around its metal-ion vertices. The acid stability of 1 was found to be greater than that of the analogous tetrahedron 2 built from 2-formylpyridine. Intriguingly, the peripheral presence of additional pyridine rings in 1 resulted in distinct guest binding behavior from that of 2, affecting guest scope as well as binding affinities. The different stabilities and guest affinities of capsules 1 and 2 enabled the design of systems whereby different cargoes could be moved between cages using acid and base as chemical stimuli.
Project description:A series of aromatic isonitriles were synthesized and their binding to sheep haemoglobin and horse heart myoglobin was investigated. The disubstituted ligands 2,6-dimethylphenylisonitrile and 2,6-diethylphenylisonitrile were found to bind to horse-heart myoglobin with affinities ranging from 500 to 5000 times greater than that of ethylisonitrile (4.6 x 10(-6) M) which has been the tightest binding isonitrile ligand for myoglobin thus far reported. The tight binding was not found to vary significantly with pH or temperature. An explanation for the unexpectedly high affinity is offered in terms of the electronic structure of aromatic isonitriles.
Project description: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.