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T C Jenkins

Publications and source records attributed to T C Jenkins.

At least 55 records · Page 3Linked to original sources
PubMed1999

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.

Acid Phosphatase↗
PubMed1999

Stabilization of DNA triple helices by a series of mono- and disubstituted amidoanthraquinones.

We have used quantitative DNase I footprinting to measure the relative affinities of four disubstituted and two monosubstituted amidoanthraquinone compounds for intermolecular DNA triplexes, and have examined how the position of the attached base-functionalized substituents affects their ability to stabilize DNA triplexes. All four isomeric disubstituted derivatives examined stabilize DNA triplexes at micromolar or lower concentrations. Of the compounds studied the 2,7-disubstituted amidoanthraquinone displayed the greatest triplex affinity. The order of triplex affinity for the other disubstituted ligands decreases in the order 2,7 > 1,8 = 1,5 > 2,6, with the equivalent monosubstituted compounds being at least an order of magnitude less efficient. The 1,5-disubstituted derivative also shows some interaction with duplex DNA. These results have been confirmed by molecular modelling studies, which provide a rational basis for the structure-activity relationships. These suggest that, although all of the compounds bind through an intercalative mode, the 2,6, 2,7 and 1,5 disubstituted isomers bind with their two side groups occupying adjacent triplex grooves, in contrast with the 1,8 isomer which is positioned with both side groups in the same triplex groove.

Amides↗
PubMed1999

Lactation performance and fatty acid composition of milk from Holstein cows fed 0 to 5% oleamide.

Diets containing 0 to 5% oleamide were fed to Holstein cows to determine linear or nonlinear responses to the fat supplement on lactation performance and milk fatty acid composition. Six rations containing concentrate, corn silage, and 0, 1, 2, 3, 4, or 5% (dry matter basis) added oleamide were fed to six multiparous cows in a 6 x 6 Latin square for 2-wk periods. As the oleamide concentration in the ration increased from 0 to 5%, dry matter intake declined, fiber and dry matter digestibilities remained constant, and digestibilities of protein and fatty acids increased. Milk yield declined as dietary oleamide increased, although yield was not depressed numerically until oleamide exceeded 2% of the diet dry matter. The C18:1 concentration doubled in milk as oleamide in the diet increased from 0 to 5%. Ratios of C18:1 to C16:0 in milk fat were 0.56, 0.83, 1.34, 1.53, and 1.73 for the diets supplemented with 0, 1, 2, 3, 4, and 5% oleamide, respectively. No amide was detected in milk samples taken from cows fed the 5% oleamide diet. Results show that intake of diets containing 2 to 3% oleamide substantially increased the milk C18:1:C16:0 ratio without greatly affecting milk yield or causing detectable amounts of amide in milk.

Animal Feed↗
PubMed1999

Interaction of tallow and hay particle size on ruminal parameters.

Four nonlactating ruminally cannulated Holstein cows were used in a 4 x 4 Latin square experiment with 4 21-d periods to determine if the effects of dietary fat would be affected by hay particle length. Treatments consisted of two levels of tallow (0 and 5%) and two hay particle lengths (short-cut and long-cut) in a 2 x 2 factorial. Diets contained alfalfa hay, corn silage, and concentrate [1:1:2, dry matter (DM) basis] fed as a total mixed ration (TMR) once per day. Samples of the 0 and 5% tallow TMR were ground and incubated in situ in polyester bags for 24 and 48 h. Ruminal samples were taken on day 21 at 0800 h and at 2-h intervals until 1600 h. The total tract digestibilities of acid detergent fiber (ADF) and neutral detergent fiber (NDF) were not affected by tallow or by hay by tallow interactions. There was a trend for tallow to improve total tract digestibility of crude protein (CP) (70.2 vs. 74.7%). After 48 h of ruminal incubation, tallow significantly decreased the digestibilities of DM, ADF, and NDF. No hay length by tallow interactions for DM, NDF, ADF or CP digestibilities occurred after 24 or 48 h. Tallow increased concentrations of propionate and decreased concentrations of acetate and valerate and the acetate-to-propionate ratio. Total volatile fatty acids increased when tallow was added to diets with short-cut hay, which suggests that when unprotected fat is added to diets with a high level of hay, a short-cut hay length may be advantageous. This result may be due to shorter rumen retention time of feed particles, which reduces the time for fatty acids to exert antimicrobial effects. Or, it may because the increased surface area of the hay particle provides more area for microbial attachment and increased fermentation.

Adipose Tissue↗
PubMed1998

Human telomerase inhibition by regioisomeric disubstituted amidoanthracene-9,10-diones.

Telomerase is an attractive target for the design of new anticancer drugs. We have previously described a series of 1,4- and 2, 6-difunctionalized amidoanthracene-9,10-diones that inhibit human telomerase via stabilization of telomeric G-quadruplex structures. The present study details the preparation of three further, distinct series of regioisomeric difunctionalized amidoanthracene-9,10-diones substituted at the 1,5-, 1,8-, and 2,7-positions, respectively. Their in vitro cytotoxicity and Taq DNA polymerase and human telomerase inhibition properties are reported and compared with those of their 1,4- and 2,6-isomers. Potent telomerase inhibition (telIC50 values 1.3-17.3 microM) is exhibited within each isomeric series. In addition, biophysical and molecular modeling studies have been conducted to examine binding to the target G-quadruplex structure formed by the folding of telomeric DNA. These studies indicate that the isomeric diamidoanthracene-9,10-diones bind to the human telomeric G-quadruplex structure with a stoichiometry of 1:1. Plausible G-quadruplex-ligand complexes have been identified for each isomeric family, with three distinct modes of intercalative binding being proposed. The exact mode of intercalative binding is dictated by the positional placement of substituent side chains. Furthermore, in contrast to previous studies directed toward triplex DNA, it is evident that stringent control over positional attachment of substituents is not a necessity for effective telomerase inhibition.

Anthracenes↗
PubMed1998

Thermodynamic, kinetic, and conformational properties of a parallel intermolecular DNA triplex containing 5' and 3' junctions.

The interaction of the 11-mer oligodeoxypyrimidine d(TCTTCTUTCCT) with the 17 bp duplex d(CGCTAGAAGAAAGGACG).d(CGTCCUTTCTTCTAGCG) in forming an intermolecular DNA triplex has been examined in solution by surface plasmon resonance (SPR), UV thermal denaturation, circular dichroism (CD), and NMR methods. Thermodynamic data were also acquired for the shorter 15 bp target duplex d(CGCTAGAAGAAAGGA). d(TCCUTTCTTCTAGCG), which forms a 3' flush-ended parallel triplex. CD titrations at pH 5 gave a triplex --> (duplex + strand) dissociation constant Kd of 0.5 microM at 15 degreesC and approximately 2 microM at 25 degreesC for both the 11-15.15 and 11-17.17 systems, in agreement with analysis of the UV melting data and a direct calorimetric measurement. In contrast, the "apparent" Kd value determined by SPR was 10-20-fold smaller. The rate constant for dissociation (kd) of the third strand from the triplex was found to be approximately 0.0002 s-1 at 25 degreesC by SPR. The rate constant for exchange between the triplex and duplex states determined by NMR was approximately 2 s-1 at 40 degreesC. The dissociation kinetics measured by SPR are considerably underestimated, which largely accounts for the poor estimation of Kd using this technique. Extensive 1H NMR assignments were obtained for both the 17 bp DNA duplex and the triplex. Large changes in chemical shifts were observed in the purine strand of the host duplex, but only small shift changes were induced in the complementary pyrimidine strand. Dramatic differences in shifts were observed for the G and A residues, especially in the minor groove, consistent with only small, localized conformational changes in the underlying duplex. The magnitude of the shift changes decreased to baseline within one base of the 3' triplex-duplex junction and over two to three bases at the 5' junction. Chemical shift changes at the 5' junction suggest small conformational anomalies at this site. COSY and NOESY spectra indicate that the nucleotides are in the "S" domain in both the triplex and duplex states. These data rule out major conformation changes at the triplex-duplex boundaries. NOEs between pyrimidines in the third strand and those in the duplex showed proximity for these bases in the major groove, which could be ascribed to buckling of the Hoogsteen bases out of the plane of the Watson-Crick base pairs.

Base Pairing↗
PubMed1998

Anthracene-9,10-diones as potential anticancer agents: bacterial mutation studies of amido-substituted derivatives reveal an unexpected lack of mutagenicity.

Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254. Six of the compounds were also tested in S. typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls. Mitoxantrone was mutagenic to S. typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant. By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined. Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.

Animals↗
PubMed1998

1,4- and 2,6-disubstituted amidoanthracene-9,10-dione derivatives as inhibitors of human telomerase.

A number of 1,4- and 2,6-difunctionalized amidoanthracene-9, 10-diones have been prepared. We have examined their in vitro cytotoxicity in several tumor cell lines and their ability to inhibit the telomere-addition function of the human telomerase enzyme together with their inhibition of the Taq polymerase enzyme. Compounds with -(CH2)2- side chains terminating in basic groups such as piperidine show inhibition of telomerase at telIC50 levels of 4-11 microM. These are thus among the most potent nonnucleoside telomerase inhibitors reported to date. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Implications for amidoanthracene-9,10-dione telomerase inhibitors as potential anticancer agents are discussed.

Anthraquinones↗
PubMed1998

Influence of soybean oil in high fiber diets fed to nonlactating cows on ruminal unsaturated fatty acids and nutrient digestibility.

An experiment with a 5 x 5 Latin square design was conducted to determine the effects of the addition of soybean oil to high fiber diets on ruminal fermentation and ruminal lipid concentrations. Diets were 50% bermudagrass hay and 50% concentrate. Soybean oil was added to diets at 0, 2, 4, 6, or 8% of the dietary dry matter (DM). Ruminal samples were collected every 2 h on the last day of each period and analyzed for volatile fatty acids and lipids. The addition of soybean oil decreased DM and organic matter intake but increased fatty acid intake. Soybean oil had no effect on total tract digestibility of DM, organic matter, N, or neutral detergent fiber but decreased digestibility of fatty acids. The addition of soybean oil decreased total volatile fatty acid concentrations and the acetate to propionate ratio. Ruminal concentrations of unsaturated free fatty acids increased nonlinearly as soybean oil in the diets increased but remained < 0.67 mg/g of DM or 3% of the total fatty acids. Ruminal concentrations of total fatty acids and total saturated fatty acids increased nonlinearly as soybean oil in the diets increased. Total unsaturated fatty acid concentrations increased linearly as soybean oil increased. Neutral lipid concentrations in the rumen did not respond to increased soybean oil. These data indicate that large amounts of soybean oil can be fed in high fiber diets without greatly increasing the concentration of ruminal unsaturated fatty acids or depressing nutrient digestibility.

Animal Nutritional Physiological Phenomena↗
PubMed1998

Fatty acid composition of milk from Holstein cows fed oleamide or canola oil.

Based on previous in vitro results that showed reduced biohydrogenation of oleamide by ruminal microbes, this study was conducted to determine whether the addition of oleamide to the diets of dairy cows would enhance the C18:1 concentration in milk. Nine first lactation Holstein cows were fed three diets in a 3 x 3 Latin square replicated three times. Each period lasted 3 wk. The total mixed diets consisted of 42% corn silage and 58% concentrate (dry matter basis) with either no added fat (control), 3.5% high oleic canola oil, or 3.5% oleamide. Dry matter intake was reduced when oleamide was added to the diet but not when canola oil was added. Milk yields were the same for cows fed all three diets. Canola oil reduced fat-corrected milk yield and milk fat concentration, but these were not affected by oleamide. Milk protein concentration was lower for cows fed oleamide than for cows fed canola oil. Milk C18:1 averaged 23.16% of total fatty acids for cows fed the control diet and increased to 35.13% when canola oil was fed. Oleamide further increased C18:1 to 48.16% of total fatty acids in milk. All fatty acids with > or = 16 carbon chain length were reduced by oleamide. Oleamide was more effective than was canola oil in this study at increasing the oleic acid content of bovine milk. Oleamide reduced dietary intake when added at 3.5% of the dietary dry matter but still had no effect on milk yield or milk composition.

Animals↗
PubMed1998

Interactions of tallow and hay particle size on yield and composition of milk from lactating Holstein cows.

An 18-wk lactation study was conducted to determine whether the effects of tallow on the lactation performance of dairy cows were influenced by particle size of hay in the ration. A total mixed ration containing 50% concentrate, 25% corn silage, and 25% alfalfa hay (dry matter basis) was fed to Holstein cows. Four total mixed rations were developed based on differences in the percentage of tallow in the concentrate and particle size of alfalfa hay: 1) 0% tallow, long-cut hay; 2) 0% tallow, short-cut hay; 3) 5% tallow, long-cut hay; and 4) 5% tallow, short-cut hay. Ration had no effect on dry matter intake, body weight gain or change in body condition score. Tallow increased milk and milk protein yields but reduced milk protein concentration. However, the effects of tallow on milk and milk protein yields were the same, regardless of hay length in the ration. A tendency for an interaction of tallow and hay particle size was detected for fat-corrected milk (FCM) because tallow increased FCM more when hay was short. Ration had no effect on volatile fatty acids in ruminal samples collected via a stomach tube. In this study, the effects of tallow on milk yield and composition from Holstein cows were the same, regardless of hay particle size in the ration. The tendency for tallow to increase FCM more when hay was short suggests at least a limited role of forage particle size in the determination of how fat supplements in dairy rations affect lactation performance.

Animal Feed↗
PubMed1998

Effect of ruminally protected amino acids on milk yield and composition of Jersey cows fed whole cottonseed.

The objectives of this experiment were to determine whether ruminally protected amino acids (AA) increased milk protein when this content was depressed by the addition of whole cottonseeds in the diets of early lactation Jersey cows. Treatments were 1) a control diet, 2) a diet containing whole cottonseed, and 3) a diet containing whole cottonseed and ruminally protected lysine and methionine. Cows were assigned to treatments at a mean of 7 d postpartum and remained on the experiment for 18 wk. Dry matter intake and yields of milk, milk fat, fat-corrected milk, and energy-corrected milk were not affected by treatment. Milk fat content tended to decrease for cows fed diets containing whole cottonseed. However, the percentages of milk protein, total N, and casein N were depressed by the addition of whole cottonseed and were increased by the addition of ruminally protected AA. Plasma concentrations of methionine, but not lysine, were increased when ruminally protected AA were fed, suggesting that lysine was the most limiting.

Amino Acids↗
PubMed1997

1-[(omega-aminoalkyl)amino]-4-[N-(omega-aminoalkyl)carbamoyl]-9-oxo-9, 10-dihydroacridines as intercalating cytotoxic agents: synthesis, DNA binding, and biological evaluation.

A series of DNA-intercalating potential antitumor agents, 1-[(omega-aminoalkyl)amino]-4-[N-(omega-aminoalkyl)carbamoyl]-9-oxo-9, 10-dihydroacridines, has been prepared by aminolysis of the corresponding 4-[N-(omega-aminoalkyl)carbamoyl]-1-chloro derivative with a suitable omega-aminoalkylamine. The noncovalent DNA-binding properties of these bis-functionalized compounds have been examined using a combination of fluorometric and thermal denaturation techniques and are compared with the behaviors for established DNA intercalants and cationic minor groove ligands. The results indicate that (i) the agents are considerably more DNA-affinic than less functionalized acridinones, with 'apparent' binding constants of (0.1-2.1) x 10(7) and (0.3-7.5) x 10(7) M-1 at pH 5 and 7, respectively, (ii) overall affinity is sensitive to both the length of the flexible side chain and the complexity of the attached amine substituents, and (iii) the pendant side chains effect a switch to moderate AT-preferential binding. In vitro cytotoxic potencies toward six tumor cell lines broadly parallel the observed DNA affinities, although poor correlation is evident for certain compounds. The octanol/water partition coefficients have been also calculated, but there is no correlation with cytotoxicity values. Two highly DNA-affinic analogs, 10 and 13, have been identified with a useful broad spectrum of cytotoxic activity.

Acridines↗
PubMed1997

Specific binding of hoechst 33258 to the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies.

Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry (ITC) techniques have been used to examine the binding characteristics of Hoechst 33258 with the extended AT-tract DNA duplex d(CGCAAATTTGCG)2 in aqueous solution. The method of continuous variation reveals a 1:1 binding stoichiometry. Fluorescence equilibrium studies carried out at three different, but fixed, ligand concentrations show that the binding isotherm shifts towards higher [DNA] as the concentration of ligand is increased. The data show tight binding with Kb=3.2(+/-0.6)x10(8) M(duplex)-1 at 25 degrees C in solutions containing 200 mM Na+. Based on UV studies of duplex melting, which show that strand separation starts at approximately 35 degrees C and has a Tm at 54 degrees C in 300 mM NaCl, binding enthalpies were determined by ITC in the 10 to 30 degrees C range. Binding is endothermic at all temperatures examined, with DeltaH values ranging from +10.24(+/-0.18) to +4.2(+/-0.10) kcal mol(duplex)-1 at 9.4 degrees C and 30.1 degrees C, indicating that the interaction is entropically driven. The temperature dependence of DeltaH shows a binding-induced change in heat capacity (DeltaCp) of -330(+/-50) cal mol-1 K-1. This value is similar to that predicted from a consideration of the effects of hydrophobic and hydrophilic solvent-accessible surface burial on complexation. This result, almost entirely dictated by a removal from exposure of the non-polar reactant surfaces, represents the first demonstration of such behavior in a DNA-drug system. The salt dependence of the binding constant was examined using reverse-salt fluorescence titrations, with a value of 0.99 determined for the deltalnK/deltaln[Na+] parameter. These data provide a detailed thermodynamic profile for the interaction that enables a dissection of DeltaGobs into the component free energy terms. Analysis of data obtained at 25 degrees C reveals that DeltaGobs is dominated by the free energy for hydrophobic transfer of ligand from solution to the DNA binding site. Molecular interactions, including H-bonding and van der Waals contacts, are found to play only a minor role in stabilizing the resulting complex, a somewhat surprising finding given the emphasis placed on such interactions from structural studies.

Base Sequence↗
PubMed1997

Inhibition of bacteriophage T7 RNA polymerase in vitro transcription by DNA-binding pyrrolo[2,1-c][1,4]benzodiazepines.

The interactions of several pyrrolo[2, 1-c][1,4]benzodiazepine (PBD) antitumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been analyzed by quantitative in vitro transcription (QIVT) and in vitro transcription footprinting (IVTF) methods. A concentration-dependent inhibitory effect of the PBDs on transcription is observed using both techniques. The rank order for overall inhibition of transcription by the QIVT method is found to be: sibiromycin > tomaymycin > anthramycin > DC-81 > neothramycin, whereas the IVTF experiments show a different ranking: sibiromycin > anthramycin > neothramycin > tomaymycin. In addition, stimulation of transcription was observed at low PBD concentrations in both the QIVT and IVTF experiments. These results demonstrate unequivocally that the formation of PBD-DNA adducts at AGA-5' base sequences on the transcribed strand results in transcription blockage for all PBDs examined. Furthermore, the sequence of flanking base pairs appears to influence the degree of blocking, with the sequences ACAGAAA-5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounced blocks for all PBDs studied in this system. Neothramycin and tomaymycin cause additional blocks at some GGA-5' and TGA-5' sequences. Parallel MPE-Fe(II) footprinting studies have revealed PBD binding sites on both the transcribing and nontranscribing strands, although all transcription blocks determined from the IVTF assays are due to drug bound on the transcribing DNA template strand.

Anthramycin↗
PubMed1997

AT selectivity and DNA minor groove binding: modelling, NMR and structural studies of the interactions of propamidine and pentamidine with d(CGCGAATTCGCG)2.

A molecular modelling strategy has been developed to identify potential binding sites for bis(amidine) ligands in the minor groove of duplex DNA. Calculations of interaction energy for propamidine and pentamidine with d(CGCGAAT TCGCG)2 show that this duplex contains two symmetrically equivalent binding sites of identical affinity, each displaced by 0.3-0.4 bp from the centre of the AT segment. The ligands occupy groove sites spanning approximately 4 and 4-5 bp, respectively with asymmetric binding to the 5'-AATT sequence. The DNA-bis(amidine) interactions have been examined by high-resolution 1H-NMR. The patterns of induced changes in DNA proton chemical shift and the DNA-ligand NOEs confirm that both agents bind in the AT minor groove in a non-centrosymmetric fashion. Detailed structures were determined for each complex using a NOE-restrained simulated annealing procedure, showing that the B-type DNA conformation is not significantly altered upon complexation with either ligand. The free DNA duplex has previously been shown to be extensively hydrated in the minor groove [Kubinec, M.G. and Wemmer, D.E. (1992) J. Am, Chem. Soc. 114, 8739-8740 Liepinsh, E. Otting, G. and Wüthrich, K. (1992) Nucleic Acids Res. 20. 6549-6553]. We detect hydration water close to the A(H2) protons in the presence of propamidine, which may stabilise certain waters against exchange. This conclusion supports recent crystallographic analyses, suggesting that such ligands may use water molecules to bridge between amidinium protons and host DNA bases Details of the ligand interactions with AT-tract DNA duplexes can now be compared for the subsequences 5'-AAT, 5'-AATT and 5'-AAATTT.

Base Sequence↗
PubMed1997

Hydration and solution structure of d(CGCAAATTTGCG)2 and its complex with propamidine from NMR and molecular modelling.

The hydration of the d(CGCAAATTTGCG)2 duplex and its complex with a propamidine reporter ligand has been examined in aqueous solution by two-dimensional NMR at two spectrometer frequencies and three temperatures. Quantitative analysis of ROESY and NOESY cross-peaks showed effective correlation times of approximately 0.5 ns at 283 K for DNA-water interactions in the major groove. In some cases the sign of the NOE inverts on changing either the temperature or spectrometer frequency. Larger effective correlation times of approximately 1 ns were observed for water interactions with A5(H2) and A6(H2) atoms located in the minor groove. Interproton NOEs and changes in chemical shifts showed that propamidine binds in the minor groove 5'-AATTT region of the host duplex, but does not displace waters adjacent to either A5(H2) or A6(H2). In the complex, the effective correlation times of these waters increase more than two-fold, possibly as a result of stabilisation due to H-bonded interaction with the amidine groups of the ligand. Hydration of the bound molecule was also found, suggesting that water may contribute to the DNA binding process for bis(amidine) drugs. Structure refinement by a NOE-restrained dynamic annealing procedure revealed that ligand binding is non-centrosymmetric with respect to the duplex, in accordance with the energetically favoured 5'-ATT (= 5'-AAT) sites predicted by analytical molecular modelling. In particular, the bound propamidine spans 3-4 base pairs in the A6-T7-T8 tract and makes close H-bonded contacts with A(N3/O4) acceptors positioned close to the minor groove floor. The refined NMR structure for the DNA-propamidine complex is compared with that determined recently using X-ray crystallographic methods.

Base Sequence↗