Biospecific Interaction Analysis (BIA) of Low-Molecular Weight DNA-Binding Drugs (original) (raw)
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European Journal of Pharmacology: Molecular Pharmacology, 1995
Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we studied sequence-selectivity of the binding of chromomycin and distamycin to DNA by performing DNase I footprinting and analysis of the cleaved fragments by the Pharmacia ALF TM DNA Sequencing System. As a model system we employed the long terminal repeat of the human immunodeficiency type 1 virus. The main conclusion of our experiments is that automated analysis of DNase I footprinting is a fast and reliable technique to study drugs-DNA interactions. The results obtained suggest that distamycin and chromomycin differentially interact with the long terminal repeat of the human immunodeficiency type 1 virus; this differential binding depends upon the DNA sequences recognized. The data presented are consistent with a preferential binding of distamycin to DNA sequences of the binding sites of nuclear factor kappa B and transcription factor IID. By contrast, distamycin exhibits only weak binding to DNA sequences recognized by the promoter-specific transcription factor Spl. Unlike distamycin, chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Spl.
International Journal of Oncology, 1997
DNA-binding drugs interfere with the activity of a large variety of transcription factors, leading to an alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases. The analysis of sequence selectivity of DNA-binding drugs by footprinting, gel retardation studies, polymerase chain reaction and in vitro transcription does not allow an easy study of kinetics of binding and dissociation. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables the monitoring of a variety of molecular reactions in realtime by surface plasmon resonance (SPR). In this report we demonstrate that molecular interactions between the DNAbinding drug chromomycin and a biotinylated GC-rich Ha-ras oligonucleotide probe immobilized on a sensor chip is detectable by SPR technology using the BIAcore™ biosensor. This approach appears of interest in the development of drugs exhibiting differential affinity for target DNA sequences for the following reasons: a) results are obtained within one hour; b) unlike footprinting and gel retardation studies, this technology does not require 32 P-labelled probes; c) BIA allows kinetic studies of both association and dissociation.
Current Drug Targets - Immune, Endocrine & Metabolic Disorders, 2005
The experimental determination of the binding constant of a drug for its target molecule is of considerable importance. It is a basic experimental parameter in a variety of studies, such as the prediction of drug efficiency, or in the pharmacokinetic drug interaction. DNA-binding drugs have been reported to be able to interfere in a sequence dependent manner with biological functions such as topoisomerase activity, restriction of enzyme cleavage of DNA, protein-DNA interactions and the activity of transcription factors, leading to alteration of gene expression. This effect could have important practical application in the experimental therapy of human pathologies, including neoplastic diseases and viral, or microbial infections. The assessment of the biological activity of DNA-binding drugs by polymerase chain reaction, footprinting, gel retardation and in vitro transcription studies was recently reported. However, most of these techniques are steady-state methodologies and therefore are not suitable for an easy determination of the binding activity of DNAbinding drugs to target DNA and the stability of drugs-DNA complexes. Direct real-time observation and measurement of the interaction between DNA-binding drug and target DNA sequence is a subject of interest for drug discovery and development. The recent development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions of DNA-binding drugs with target DNA elements in real-time. The present review is designed to indicate the theoretical background of SPR-based biosensor technology as well as to present the great variety of measurements and modes of interaction kinetics that can be performed with these techniques. In addition, some of the most recent studies in determining the binding constant and stoichiometry of DNA-binding drugs to target DNA with SPR technology are reviewed and the available theoretical aspects necessary for the comprehension of the experiments are provided.
Nucleic Acids Research, 1979
Interaction of DNA with the analogs of the antibiotic distazycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The bindin isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to po1y(dA)-poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GO pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA.
On the kinetics of distamycin binding to its target sites on duplex DNA
Proceedings of the National Academy of Sciences, 2000
Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamycin binding reaction has not been studied in either of its binding modes, because of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant increase in fluorescence amplitude, accompanied by a small red shift, on binding distamycin to its specific target sites. This signal can be used to monitor drug binding in steady-state and timeresolved processes. Distamycin shows extremely fast association with the 1:1 binding site, with a bimolecular rate of 7 ؋ 10 7 M ؊1 ⅐s ؊1 and also fairly rapid dissociation (Ϸ3 s ؊1). When DNA is in excess, there is a slow component in the association reaction whose rate decreases strongly with increasing DNA concentration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecular rate comparable to that at the 1:1 site, followed by a slow (Ϸ4 s ؊1) equilibration to the final population. Dissociation from the 2:1 site is Ϸ40-fold slower than from the 1:1 site. This study provides the groundwork for analysis of the binding kinetics of longer polyamides and covalently linked polyamides that have recently been shown to inhibit transcription in vivo.
Chemistry and biology of DNA-binding small molecules
2012
Regulation of the transcription machinery is one of the many ways to achieve control of gene expression. This has been done either at the transcription initiation stage or at the elongation stage. Different methodologies are known to inhibit transcription initiation via targeting of double-stranded (ds) DNA by: (i) synthetic oligonucleotides, (ii) ds-DNA-specific, sequenceselective minor-groove binders (distamycin A), intercalators (daunomycin) combilexins and (iii) small molecule (peptide or intercalator)-oligonucleotide conjugates. In some cases, instead of ds-DNA, higher order G-quadruplex structures are formed at the start site of transcription. In this regard G-quadruplex DNA-specific small molecules play a significant role towards inhibition of the transcription machinery. Different types of designer DNA-binding agents act as powerful sequence-specific gene modulators, by exerting their effect from transcription regulation to gene modification. But most of these chemotherapeutic agents have serious side effects. Accordingly, there is always a challenge to design such DNA-binding molecules that should not only achieve maximum specific DNA-binding affinity, and cellular and nuclear transport activity, but also would not interfere with the functions of normal cells.
Designing DNA Binding Antitumor Antibiotics With Structure Determination: A Systems Approach
2008
Hoechst-33258 was designed in order to furnish GC-sequence binding drugs. By closely analyzing the DNA binding structural parameters needed in the structure of a drug, it was effectively concluded that by replacing C-atoms with a more electronegative atoms like N atoms would afford the change in DNA sequence specificity of the DNA minor groove binding antitumor compounds. The structural analysis of modified drug: DNA complexes has revealed the change of DNA sequence specificity from AT to GC. In another case, the derivatives of Hoechst-33258 with N or O-atom at selected positions resulted in the GCsequence selective DNA binding. With systems approach, a few selectively chosen C-atoms in a pyrrole or benzimidazole rings were replaced with H-bond donor atoms like O or N atoms. This DNA sequence specificity has also resulted in the enhanced DNA topoisomerase inhibiting profile, needed for antitumor activity.
Journal of Molecular Modeling, 2007
A 3D-QSAR analysis has been carried out by comparative molecular field analysis (CoMFA) on a series of distamycin analogs that bind to the DNA of drug-resistant bacterial strains MRSA, PRSP and VSEF. The structures of the molecules were derived from the X-ray structure of distamycin bound to DNA and were aligned using the Database alignment method in Sybyl. Statistically significant CoMFA models for each activity were generated. The CoMFA contours throw light on the structure activity relationship (SAR) and help to identify novel features that can be incorporated into the distamycin framework to improve the activity. Common contours have been gleaned from the three models to construct a unified model that explains the steric and electrostatic requirements for antimicrobial activity against the three resistant strains. Figure A unified CoMFA model for broad-spectrum DNA minor-groove binders
Label-Free Monitoring of DNA–Ligand Interactions
Analytical Biochemistry, 1997
sess, e.g., anti-tumor, anti-viral, or anti-microbial activ-We report on the label-and isotope-free monitoring ity, and certain substances are of pharmacological and of DNA interactions with low-molecular-weight limedical importance (2). Many anti-tumor drugs exert gands. An optical technique based on interference at their action by interfering with the function of DNA. thin layers was used to monitor in real time binding Modes of interaction include noncovalent binding such as of ligands at DNA which was immobilized by Coulomb intercalation and groove binding (1), drug-DNA adduct interactions at a positively charged surface. Approxiformation, e.g., by cisplatin covalent binding , and mately 2 ng DNA/mm 2 was irreversibly bound to the DNA backbone scission, e.g., by bleomycin or enediyne surface, which remained stable over several days. This antibiotics (4, 5). Anthracycline antibiotics constitute an result was confirmed by characterization of the layer important family of intercalative anti-tumor drugs and using spectroscopic ellipsometry. During incubation some of them, e.g., doxorubicin (6), have been used cliniof immobilized DNA with a variety of intercalators and cally as components in chemotherapeutic treatments of other DNA-binding compounds in a flow system, interdifferent kinds of cancer. Intercalators bind to DNA by actions were monitored by reflectometric interference inserting their planar chromophores between adjacent spectroscopy. Binding effects between 10 and 400 pg/ DNA base pairs. Frequently, these complexes are further mm 2 were detected unambiguously. Nonspecific bindstabilized by hydrogen bond formation between the DNA ing effects were excluded by using a negatively bases and the sugar moieties appended to the aglycon.