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D E Graves

Publications and source records attributed to D E Graves.

At least 37 records · Page 2Linked to original sources
PubMed1993

Influence of DNA base sequence on the binding energetics of actinomycin D.

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.

Amino Acid Sequence↗
PubMed1992

Structure-activity studies of amsacrine analogs in drug resistant human leukemia cell lines expressing either altered DNA topoisomerase II or P-glycoprotein.

In an attempt to characterize and overcome tumor cell resistance to amsacrine (m-AMSA), we studied the structure-activity relationships for amsacrine and seven of its analogs. Using the human leukemic cell line, CCRF-CEM, and its derivatives that express either P-glycoprotein (Pgp)-associated multidrug resistance (MDR) (CEM/VLB100) or altered topoisomerase II-associated MDR (at-MDR) (CEM/VM-1), we assessed antitumor effects of these drugs in a 48-hr growth inhibition assay. We also measured drug-topoisomerase II interactions in an intact cell assay that permits quantitation of drug-stabilized DNA-topoisomerase II complexes. We found that among the tested compounds, amsacrine has an intermediate effect on cell growth in all three cell lines. The CEM/VM-1 cells were 8.6-fold cross-resistant to m-AMSA, and the cross-resistance to the analogs was from 3.0- to 10.5-fold. In the CEM/VLB100 cells, the resistance pattern was different: several analogs, including amsacrine, showed little or no cross-resistance (0.5- to 2.8-fold), whereas for those compounds with substituents at position 3 on the acridine ring, resistance was relatively higher (9.9- or 16.2-fold). Substituents at this position substantially decrease the lipophilicity of the two compounds examined, probably because they both contain amino groups that would be charged at physiologic pH. Compound 12489, having a 1'-NHSO2C6H4NH2 substituent, was very potent in the three cell lines, showing only a slightly higher IC50 value in the CEM/VM-1 line and a lower IC50 value in the CEM/VLB100 and in the CEM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies.

The effects of covalent modification of poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) by ethidium monoazide (a photoreactive analogue of ethidium) on the salt-induced B to Z transition are examined. Earlier studies have shown ethidium monoazide to bind DNA (in the absence of light) in a manner identical to that of the parent ethidium bromide. Photolysis of the ethidium monoazide-DNA complex with visible light results in the covalent attachment of the photoreactive analogue to the DNA. This ability to form a covalent adduct was utilized to probe the effects of an intercalating irreversibly bound adduct on the salt-induced B to Z transition of the poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) polynucleotides. In the absence of drug, the salt-induced transition from the B to Z structure occurs in a highly cooperative manner. In contrast, this cooperativity is diminished as the concentration of covalently attached drug is increased. The degree of inhibition of the B to Z transition is quantitated as a function of the concentration of covalently attached drug. At a concentration of one drug bound per four base pairs for poly(dGdC).poly(dGdC) and seven base pairs for poly(dGm5dC).poly(dGm5dC), total inhibition of this transition is achieved. Lower concentrations of bound drug were effective in the partial inhibition of this transition. The effects of the covalently bound intercalator on the energetics of the B to Z transition were determined and demonstrated that the adduct is effective in locking the alternating copolymer in a right-handed conformation under high salt conditions.

Calorimetry↗
PubMed1991

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies.

The photoaffinity analogue ethidium monoazide was used to prepare samples of poly(dGdC).poly(dGdC) containing covalently attached ethidium. The effects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly(dGdC).poly(dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC)4, (dGdC)5, and (dGdC)6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2 +/- 1.1 kcal mol-1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumably by locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.

Affinity Labels↗
PubMed1991

Interactions of anilinoacridines with nucleic acids: effects of substituent modifications on DNA-binding properties.

Spectroscopic methods are used to probe the interactions of several anilinoacridine analogues with calf thymus DNA over a wide range of temperatures and sodium chloride concentrations. The structurally similar compounds m-AMSA, AMSA (both active as antitumor agents), and o-AMSA (inactive as an antitumor agent) have been widely studied in their abilities to bind DNA in an intercalative manner. Recent studies from this laboratory reveal distinct differences in the thermodynamic binding mechanisms between m-AMSA and o-AMSA (Wadkins & Graves, 1989), with the m-AMSA-DNA interaction being an enthalpy-driven process while the binding of o-AMSA to DNA is characterized by more positive entropy values. To further examine the physical chemical properties associated with these compounds and their correlation with antitumor activities, an in-depth investigation into the thermodynamic parameters of these compounds and structurally related anilinoacridine analogues was performed. These studies demonstrate that substituent type and position on the aniline ring of the anilinoacridines greatly influences both the affinities of these drugs in binding to DNA and dictates whether the DNA binding is an enthalpy- or entropy-driven process. The differences in thermodynamic mechanisms of binding between the two isomers along with molecular modeling studies reveal the electronic and/or steric factors resulting from the positioning of the methoxy substituent group on the anilino ring directs the DNA-binding properties through orientation of the methanesulfonamido group at the 1' position of the aniline ring. The orientation of this substituent group may result in favorable contacts through hydrogen bonding with neighboring base pairs and ultimately influence the biological effectiveness as an antitumor agent.

Acridines↗
PubMed1989

Two-dimensional NMR studies on the anthramycin-d(ATGCAT)2 adduct.

Two-dimensional NMR experiments were performed on the adduct of anthramycin with d(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATamGCAT).d(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOEs to several nucleotide protons. The network of anthramycin-nucleotide NOEs and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H11a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The NOE data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the J1'-2' coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred.

Anthramycin↗
PubMed1989

Thermodynamics of the interactions of m-AMSA and o-AMSA with nucleic acids: influence of ionic strength and DNA base composition.

The equilibrium binding of the antitumor agent m-AMSA and its biologically inactive analog o-AMSA to native and synthetic DNAs are compared over a wide range of ionic strengths and temperatures. Although o-AMSA binds DNA with a higher affinity than m-AMSA it is not effective as an antitumor agent. Both m-AMSA and o-AMSA bind DNA in an intercalative manner. Indepth investigations into the thermodynamic parameters of these interactions reveal the interaction of m-AMSA with DNA to be an enthalpy driven process. In contrast, the structurally similar but biologically inactive o-AMSA binds DNA through an entropy driven process. The differences in thermodynamic mechanisms of binding between the two isomers reveal that the electronic and/or steric factors resulting from the position of the methoxy substituent group on the anilino ring directs the DNA binding properties of these compounds and ultimately the biological effectiveness as an antitumor agent.

Amsacrine↗
PubMed1989

7-Azidoactinomycin D: a novel probe for examining actinomycin D-DNA interactions.

The technique of photoaffinity labeling is applied to the actinomycin D system to provide a novel probe for the examination of the interactions of actinomycin D with nucleic acids. The capacity for covalent attachment of actinomycin D will aid greatly in the study of target-site specificities and the correlations of biological effects with biophysical DNA interactions. Through chemical modification of the parent actinomycin D molecule with a photoreactive azido substituent, a functional analog of the parent actinomycin D is generated having equilibrium binding properties identical to those of the parent molecule yet with the capacity to form a covalent attachment to DNA upon photolysis. The results presented here describe the noncovalent interactions of this photoreactive probe to DNA (absence of light) and compares the binding properties observed to those of the parent actinomycin D and 7-aminoactinomycin D analog. These studies demonstrate that the DNA binding properties (i.e. binding affinity, binding site size, and sequence specificity) retained by the 7-azidoactinomycin D, thus providing a suitable probe for examining actinomycin D-DNA interactions.

Affinity Labels↗
PubMed1989

7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D.

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.

Affinity Labels↗
PubMed1988

Cooperative binding of m-AMSA to nucleic acids.

The equilibrium binding of the antitumor agent m-AMSA (4'-(9-acridinylamino) methane-sulfon-m-ansidide) has been examined by optical methods. These studies which have focused on the low bound drug concentrations (r values less than 0.02, base pairs) reveal m-AMSA to bind calf thymus DNA in a highly cooperative manner as indicated by the initial positive slope of the Scatchard plot. In contrast, the studies on the parent 9-aminoacridine under identical conditions demonstrate that this compound binds DNA in a noncooperative (neighbor exclusion) manner. The positive cooperative binding phenomenon of m-AMSA is probed as a function of ionic concentration and shown to exist over the range of salt concentrations examined (0.01 to 0.1 M); however, the magnitude of the cooperative binding is altered. This observation of cooperativity is consistent with earlier studies on biologically active compounds and may be related to such binding parameters as binding sequence selectivity and/or structural perturbations to the DNA structure.

Allosteric Site↗
PubMed1988

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxins B1 and B2 with DNA and the oligodeoxynucleotide d(ATGCAT)2.

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B1 and B2 with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B2 with the oligodeoxynucleotide d(ATGCAT)2. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B1 binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin less than 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels less than 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a "two-site" binding model [L.S. Rosenberg, M.J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002-1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B2 binding to d(ATGCAT)2 indicates that the aflatoxin B2/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1-0.5 ppm are observed for the aflatoxin B2 4-OCH3, H5, and H6a protons. Much smaller chemical shift changes (less than or equal to 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T1 experiments demonstrate a 15-25% decrease in the relaxation time for the adenine H2 protons when aflatoxin B2 is added to the solution. This result suggests that aflatoxin B2 protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B1 and B2 complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B1-guanine N7 DNA adducts.

Aflatoxin B1↗
PubMed1985

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution.

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthramycin↗
PubMed1984

The reaction of anthramycin with DNA. Proton and carbon nuclear magnetic resonance studies on the structure of the anthramycin-DNA adduct.

Nuclear magnetic resonance techniques are used to confirm the points of attachment of anthramycin to DNA. Using 13C NMR spectroscopy, the C-11 resonance of anthramycin is shown to undergo a 16-ppm upfield shift upon formation of a covalent bond with DNA, indicative of an aminal linkage at that position. The site of attachment on the DNA is determined using the self-complementary oligodeoxyribonucleotide d-(ApTpGpCpApT) as a DNA model. Proton NMR, both in H2O and D2O solutions, provides a direct characterization of the anthramycin-oligonucleotide adduct. Upon covalent attachment to the duplex, a loss in the helical symmetry is observed, resulting in a doubling of several of the oligonucleotide resonances. Examination of the data confirms that the point of attachment of the anthramycin to the d-(ApTpGpCpApT) is at the guanine-NH2-position, consistent with the model proposed by Hurley and Petrusek (Hurley, L. H., and Petrusek, R. L. (1979) Nature (Lond.) 282, 529-531) and Petrusek et al. (Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., and Hurley, L. H. (1981) Biochemistry 20, 1111-1119).

Animals↗
PubMed1983

Single-cell partition analysis--a direct fluorescence technique for examining ligand-macromolecule interactions.

Single-cell partition analysis is described as a novel technique for examining ligand-macromolecule interactions. This procedure is a combination of the classical fluorescence titration technique and phase-partition techniques and allows three separate methods for calculating and comparing both free and bound drug concentrations. The value of this technique is demonstrated by the comparison of the binding properties of the potent antitumor antibiotic adriamycin and ethidium bromide to nucleic acids. Binding isotherms of both drugs were obtained at low r (concentration of bound drug per base pair) values, showing strikingly different results, thus allowing insight to be gained into the cooperative binding of these drugs to DNA.

Animals↗
PubMed1983

Adriamycin and daunorubicin bind in a cooperative manner to deoxyribonucleic acid.

Phase partition techniques have been used to measure the binding of the antitumor drugs adriamycin (NSC-123127) and daunorubicin (NSC-82151) to various DNAs. These methods provide reliable equilibrium binding data at the low levels of drug binding that may be expected in vivo. Both adriamycin and daunorubicin exhibit positive cooperativity (and/or allosterism) in their equilibrium binding to DNA as indicated by the positive slope in the initial region of the binding isotherms (Scatchard plots) under conditions simulating physiological ionic strengths. The cooperative binding (i.e., the appearance of initial positive curvature in the binding isotherms) is dependent upon the ionic strength, which suggests a role for DNA flexibility in the cooperative binding process. An analysis of the slope of the initial portion of the binding isotherms for the interaction of adriamycin with synthetic deoxypolynucleotides shows that the degree of cooperative binding decreases in the order poly(dGdT) X poly(dAdC) greater than or equal to poly(dAdT) X poly(dAdT) greater than poly(dGdC) X poly(dGdC). Marky and Breslauer [Marky, L.A., & Breslauer, K. J. (1982) Biopolymers 21, 2185-2194] found that the average base stacking enthalpies of these synthetic poly-nucleotides were in the same order, which also suggests that the properties of the DNA influence the cooperative binding (and/or allosteric effects). Adriamycin binds with a higher degree of cooperativity than daunorubicin (0.1 M NaCl); although this correlates with the effectiveness of the drugs as antitumor agents, the exact relationship between the observation of cooperative binding and pharmacological activity is yet to be determined.

Animals↗