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Biomedical subjects

J B Chaires

Publications and source records attributed to J B Chaires.

At least 19 recordsLinked to original sources
PubMed2001

Hydration changes for DNA intercalation reactions.

The hydration changes that accompany the DNA binding of five intercalators (ethidium, propidium, proflavine, daunomycin, and 7-aminoactinomycin D) were measured by the osmotic stress method with use of the osmolytes betaine, sucrose, and triethylene glycol. Water uptake was found to accompany complex formation for all intercalators except ethidium. The difference in the number of bound water molecules between the complex and the free reactants (Deltan(w)) was different for each intercalator. The values found for Deltan(w) were the following: propidium, +6; daunomycin, +18; proflavine, +30; and 7-aminoactinomycin D, +32. For ethidium binding to DNA a value of Deltan(w) = +0.25(+/-0.3) was found, indicating that within experimental error no water was released or taken up upon complex formation. Intercalation association constants measured in D2O were found to increase relative to values measured in H2O for all compounds except ethidium. A positive correlation between the ratio of binding constants (K(D2O)/K(H2O)) and Deltan(w) was found. These combined studies identify water as an important thermodynamic participant in the formation of certain intercalation complexes.

DNA↗
PubMed2001

Analysis and interpretation of ligand-DNA binding isotherms.

Binding studies provide information of fundamental and central importance for the complete understanding of ligand-DNA interactions. Studies of ligand binding to long natural DNA samples, to synthetic deoxypolynucleotides of simple repeating sequence, and to oligonucleotides of defined sequence are all needed to begin to understand the interaction in detail. Binding studies provide entry into the thermodynamics of the DNA interactions, which in turn provides great insight into the molecular forces that drive the binding process. This chapter summarizes both model-dependent and -independent approaches for the analysis and interpretation of binding isotherms, and should serve as a concise guide for handling experimental data.

Animals↗
PubMed2001

An octakis-intercalating molecule.

Herein we report the synthesis and characterization of a polyintercalator with eight potential intercalating l,4,5,8-naphthalenetetracarboxylic diimide (NDI) units linked in a head-to-tail arrangement via a peptide linker. UV spectroscopy and viscometry measurements indicated the molecule binds to double-stranded DNA with all eight NDI units intercalated simultaneously. Competition dialysis and DNAse 1 footprinting studies revealed a preference for GC-rich regions of DNA, and circular dichroism studies revealed significant distortion of B-form DNA upon binding. Our so-called "octamer" represents, to the best of our knowledge, the first intercalator that binds as an octakis-intercalator, capable of spanning at least 16 base pairs of DNA.

Animals↗
PubMed2000

Formaldehyde-induced alkylation of a 2'-aminoglucose rebeccamycin derivative to both A.T and G.C base pairs in DNA.

Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials. Their anticancer activities are associated with their capacities to interact with DNA and to inhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazole chromophore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar residue contributes significantly to the DNA binding free energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycin derivatives in the drug-DNA complex remains poorly understood. To better understand how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on the sugar residue. We show that the presence of the 2'-amino function permits the formation of covalent drug-DNA complexes in the presence of formaldehyde. Complementary biochemical and spectroscopic measurements attest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing only A.T base pairs. The covalent binding to A.T base pairs was detected using a gel mobility shift assay and was independently confirmed by thermal denaturation studies and by fluorescence measurements using a series of synthetic polynucleotides. The HCHO-mediated alkylation reaction of the drug with A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs implicates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch grooves in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approach for probing minor versus major groove interactions between small molecules and DNA.

Alkylation↗
PubMed2000

Equilibrium unfolding of Bombyx mori glycyl-tRNA synthetase.

Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denaturant was detected as decreased fluorescence intensity and a red-shifted emission. Solute quenching of fluorescence indicated that tryptophans become progressively solvent-exposed during unfolding. Wild type enzyme had stronger negative CD bands between 220 and 230 nm than the mutant, indicative of greater alpha-helical content. Urea or guanidine-HCl caused a reduction in ellipticity at 222 nm at low denaturant concentration with the wild type enzyme, a transition that is absent in the mutant; both enzymes exhibited a cooperative transition at higher denaturant concentrations. Both enzymes dissociate to monomers in 1.5 m urea. Unfolding of wild type enzyme is described by a multistate unfolding and a parallel two state unfolding; the two-state component is absent in the mutant. Changes in spectral properties associated with unfolding were largely reversible after dilution to low denaturant. Unfolding of glycyl-tRNA synthetase is complex with a native state, a native-like monomer, partially unfolded states, and the unfolded state.

Animals↗
PubMed2000

Allosteric, chiral-selective drug binding to DNA.

The binding interactions of (-)-daunorubicin (WP900), a newly synthesized enantiomer of the anticancer drug (+)-daunorubicin, with right- and left-handed DNA, have been studied quantitatively by equilibrium dialysis, fluorescence spectroscopy, and circular dichroism. (+)-Daunorubicin binds selectively to right-handed DNA, whereas the enantiomeric WP900 ligand binds selectively to left-handed DNA. Further, binding of the enantiomeric pair to DNA is clearly chirally selective, and each of the enantiomers was found to act as an allosteric effector of DNA conformation. Under solution conditions that initially favored the left-handed conformation of [poly(dGdC)](2), (+)-daunorubicin allosterically converted the polynucleotide to a right-handed intercalated form. In contrast, under solution conditions that initially favored the right-handed conformation of [poly(dGdC)](2), WP900 converted the polynucleotide to a left-handed form. Molecular dynamics studies by using the amber force field resulted in a stereochemically feasible model for the intercalation of WP900 into left-handed DNA. The chiral selectivity observed for the DNA binding of the daunorubicin/WP900 enantiomeric pair is far greater than the selectivity previously reported for a variety of chiral metal complexes. These results open a new avenue for the rational design of potential anticancer agents that target left-handed DNA.

Allosteric Regulation↗
PubMed2000

Energetics of DNA intercalation reactions.

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.

Animals↗
PubMed2000

NB-506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA.

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])(2) was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.

Animals↗
PubMed1999

Sequence and structural selectivity of nucleic acid binding ligands.

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).

Antibiotics, Antineoplastic↗
PubMed1999

Substitution at the F-ring N-imide of the indolocarbazole antitumor drug NB-506 increases the cytotoxicity, DNA binding, and topoisomerase I inhibition activities.

The antitumor drug NB-506, which is currently undergoing phase I/II clinical trials, contains a DNA-intercalating indolocarbazole chromophore substituted with a glucose residue. In addition to interacting with DNA, the drug stabilizes the topoisomerase I-DNA covalent complex. To reinforce the DNA binding and anti-topoisomerase I activities of NB-506, an analogue containing a new substituent on the naphthalimide ring F was synthesized. The N-formylamino group of NB-506 has been replaced with a more hydrophilic group, N-bis(hydroxymethyl)methylamino. In this study we show that the incorporation of a longer substituent on the N6 position effectively reinforces both the interaction with DNA and the capacity of the drug to maintain the integrity of the topoisomerase I-DNA covalent complexes. The strength and the mode of binding of the drugs to DNA were studied by complementary biophysical techniques including absorption, fluorescence, and circular and linear dichroism. Various biochemical procedures were applied to investigate the effects on human topoisomerase I using plasmid DNA as well as restriction fragments. The drug binding sites and the positions of the topoisomerase I-mediated cleavage sites were mapped with nucleotide resolution using footprinting and sequencing techniques. Cytotoxicity measurements performed with various human cancer cell lines (HCT-116, DLD-1, MKN-45) indicate that the newly designed drug is 3 to 4 times more toxic to colon and gastric cancer cells than NB-506. Therefore, the results suggest that the antitumor activity of indolocarbazole-based drugs can be enhanced by incorporating DNA and/or topoisomerase I reactive groups. They also support the hypothesis that the substituent on the imide nitrogen on the F ring of NB-506 has direct interaction with the molecular target. The study helps to define the structure-activity relationships in the indolocarbazole series of antitumor agents targeting topoisomerase I.

Animals↗
PubMed1999

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2.

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.

Base Sequence↗
PubMed1999

Effects of hydration, ion release, and excluded volume on the melting of triplex and duplex DNA.

The stability of DNA duplex and triplex structures not only depends on molecular forces such as base pairing or tripling or electrostatic interactions but also is sensitive to its aqueous environment. This paper presents data on the melting of Escherichia coli and poly(dA).poly(dT) duplex DNA and on the poly(dT).poly(dA). poly(dT) triplex in a variety of media to assess the contributions from the osmotic status and salt content of the media. The effects of volume exclusion on the stability of the DNA structures are also studied. From thermal transition measurements in the presence of low-molecular weight osmotic stressors, the number of water molecules released upon melting is found to be four waters per base pair for duplex melting and one water for the conversion of triplex to single-strand and duplex. The effects of Na+ counterion binding are also determined in ethylene glycol solutions so that the variation of counterion binding with water activity is evaluated. The data show that there is a modest decrease in the extent of counterion binding for both duplex and triplex as water activity decreases. Finally, using larger polyethylene glycol cosolutes, the effects on melting of volume exclusion by the solutes are assessed, and the results correlated with simple geometric models for the excluded volume. These results point out that DNA stability is sensitive to important conditions in the environment of the duplex or triplex, and thus, conformation and reactivity can be influenced by these solution conditions.

DNA↗
PubMed1999

Calories from carbohydrates: energetic contribution of the carbohydrate moiety of rebeccamycin to DNA binding and the effect of its orientation on topoisomerase I inhibition.

BACKGROUND: Only a few antitumor drugs inhibit the DNA breakage-reunion reaction catalyzed by topoisomerase. One is the camptothecin derivative topotecan that has recently been used clinically. Others are the glycosylated antibiotic rebeccamycin and its synthetic analog NB-506, which is presently in phase I of clinical trials. Unlike the camptothecins, rebeccamycin-type compounds bind to DNA. We set out to elucidate the molecular basis of their interaction with duplex DNA, with particular emphasis on the role of the carbohydrate residue. RESULTS: We compared the DNA-binding and topoisomerase-I-inhibition activities of two isomers of rebeccamycin that contain a galactose residue attached to the indolocarbazole chromophore via an alpha (axial) or a beta (equatorial) glycosidic linkage. The modification of the stereochemistry of the chromophore-sugar linkage results in a marked change of the DNA-binding and topoisomerase-I- poisoning activities. The inverted configuration at the C-1' of the carbohydrate residue abolishes intercalative binding of the drug to DNA thereby drastically reducing the binding affinity. Consequently, the alpha isomer has lost the capacity to induce topoisomerase-I-mediated cleavage of DNA. Comparison with the aglycone allowed us to determine the energetic contribution of the sugar residue. CONCLUSIONS: The optimal interaction of rebeccamycin analogs with DNA is controlled to a large extent by the stereochemistry of the sugar residue. The results clarify the role of carbohydrates in stereospecific drug-DNA interactions and provide valuable information for the rational design of new rebeccamycin-type antitumor agents.

Aminoglycosides↗