Biomedical subjects
Publications and source records attributed to T C Jenkins.
A series of novel C2,C3-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via cleavage of the N10-Alloc protecting group from appropriate precursors. Biophysical and biological evaluations show that the presence of C2/C3-endo unsaturation in the PBD C-ring enhances both DNA-binding reactivity and in vitro cytotoxic potency.
A series of novel C2-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via a versatile pro-C2 ketone precursor. C2-exo-unsaturation enhances both DNA-binding reactivity and in vitro cytotoxic potency.
A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.
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.
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The design of agents targeted toward a structure-specific molecular recognition of DNA triplexes or tetraplexes ( quadruplexes ) is discussed, where such structures are relevant to antigene-based chemotherapies and the in situ cellular inhibition of telomerase function, respectively. Using principles that stem from the development of earlier synthetic duplex-binding ligands, together with recent findings that probe structure thermodynamic linkages and kinetic features of stability, a rational approach is developed to exploit the distinct molecular templates offered by these high-order nucleic acid biotarget systems. Such analytical techniques can usefully augment conventional drug design methods, particularly where detailed structural information is unavailable or the mode of binding to form a persistent DNA biotarget ligand complex is not established. Examples from the author s laboratory are used to illustrate structure-specific (or structure-preferential) recognition and subsequent stabilization of DNA triplexes using intercalative or groove-mediated binding mechanisms, and the successful targeting of DNA tetraplexes using planar extended-aromatic ligands. In each case, chemical manipulation of the molecule by exploiting either (i) geometric isomers, (ii) redistribution of charged groups and/or H-bond donors/acceptors, or (iii) optimization of intermolecular pi-overlap can be used to improve the affinity or specificity of the underlying DNA drug binding events.
Oleamide was previously reported to resist ruminal biohydrogenation and elevate milk oleic acid concentration when fed to lactating Holstein cows. To determine if Jersey cows responded similarly to oleamide, four lactating Jersey cows (mean 417 kg of body weight and 64 days in milk) were fed four diets in a 4x4 Latin square with 2-wk periods. Diets were total mixed ration containing 47% corn silage and 53% concentrate (dry matter basis) and were supplemented with no added fat (control), or with 3.5% added fat from either higholeic canola oil, a commercial source of oleamide, or oleamide synthesized from oleic acid and urea. The canola oil supplement had no effect on milk yield or composition. Compared to canola oil, the oleamide supplements reduced milk yield, dry matter intake, and milk fat and protein contents. Milk oleic acid concentration increased from 17.4% of total fatty acids for the control diet to 22.1% for the canola oil diet. Both oleamides further increased milk oleic acid to 30.0 and 27.1% of total fatty acids for the commercial and synthesized oleamides, respectively. Milk palmitic acid was reduced and stearic acid was increased by all fat supplements but more so by the oleamides than by the canola oil. Consistent with previous reports that fatty acyl amides resist ruminal biohydrogenation, feeding oleamide to Jersey cows in this study increased milk oleic acid concentration but had negative effects on feed intake and milk yield.
Six lactating Jersey cows were used in a 6 x 6 Latin square with 14-d periods to evaluate different ratios of canola oil and oleamide on nutrient digestibility, plasma fatty acids, and plasma hormones. The control diet contained no added fat. All other diets contained 3.5% added fat consisting of 0, 25, 50, 75, and 100% as oleamide and the remainder as canola oil. Data were collected during the final 4 d of each period. Dry matter intake was reduced by the addition of canola oil to the diet, and further reduced by replacing canola oil with oleamide. Milk yield was not affected by diet but increasing oleamide proportion in the fat supplement caused linear increases in cis-C18:1 and linear decreases in C4 to C16 fatty acids in milk. Adding canola oil reduced total tract digestibilities of fiber and fatty acids, but had no effect on the digestibilities of dry matter or protein. Replacing canola oil with oleamide increased protein digestibility linearly, and increased digestibility of fiber (quartic relationship) and fatty acids (quadratic relationship). Oleic acid concentration in plasma increased by adding canola oil to the diet, and was further increased by replacing canola oil with oleamide. Diet had no effect on plasma concentrations of insulin or IGF-I. Oleamide fed to Jersey cows in this study was highly digestible and had no deleterious effects on total tract digestility of fiber or protein. Increasing oleic acid concentration in plasma lipids while maintaining a constant level of added fat in the ration had no effect on circulating concentrations of insulin or IGF-I in Jerseys.
Synthetic routes have been investigated to prepare a novel C8-epoxide-functionalized pyrrolo[2,1-c][1,4]benzodiazepine 6 as a potential sequence-selective DNA cross-linking agent (Wilson et al. Tetrahedron Lett. 1995, 36, 6333-6336). A successful synthesis was accomplished via a 10-step route involving a pro-N10-Fmoc cleavage method that should have general applicability to other pyrrolobenzodiazepine (PBD) molecules containing acid- or nucleophile-sensitive groups. During the course of this work, a one-pot reductive cyclization procedure for the synthesis of PBD N10-C11 imines from nitro dimethyl acetals was also discovered, although this method results in C11a racemization which can reduce DNA binding affinity and cytotoxicity. The target epoxide 6 was shown by thermal denaturation studies to have a significantly higher DNA-binding affinity than the parent DC-81 (3) or the C8-propenoxy-PBD (15), which is structurally similar but lacks the epoxide moiety. The time course of effects upon thermal denaturation indicated a rapid initial binding phase followed by a slower phase consistent with the stepwise cross-linking of DNA observed for a difunctional agent. This was confirmed by an electrophoretic assay which demonstrated efficient induction of interstrand cross-links in plasmid DNA at concentrations >1 microM. Higher levels of interstrand cross-linking were observed at 24 h compared to 6 h incubation. A Taq polymerase stop assay indicated a preference for binding to guanine-rich sequences as predicted for bis-alkylation in the minor groove of DNA by epoxide and imine moieties. The pattern of stop sites could be partly rationalized by molecular modeling studies which suggested low-energy models to account for the observed binding behavior. The epoxide PBD 6 was shown to have significant cytotoxicity (45-60 nM) in the A2780, CH1, and CH1cis(R) human ovarian carcinoma cell lines and an IC(50) of 0.2 microM in A2780cis(R). The significant activity of 6 in the cisplatin-resistant CH1cis(R) cell line (IC(50) = 47 nM) gave a resistance factor of 0.8 compared to the parent cell line, demonstrating no cross-resistance with the major groove cross-linking agent cisplatin.
Several A-ring-modified analogues of the DNA-binding antitumor agent DC-81 (5) have been synthesized in order to study structure-reactivity/cytotoxicity relationships. For two molecules (23 and 30) the modifications required the addition of a fourth ring to give the novel dioxolo[4,5-h]- and dioxano[5,6-h]pyrrolo[2,1-c][1, 4]benzodiazepin-11-one (PBD) ring systems, respectively. Another three analogues (34, 38, and 48) have the native benzenoid A-ring replaced with pyridine, diazine, or pyrimidine rings to give the novel pyrrolo[2,1-c][1,4]pyridodiazepine, pyrrolo[2,1-c][1, 4]diazinodiazepine, and pyrrolo[2,1-c][1,4]pyrimidinodiazepine systems, respectively. The other new analogues (16a,b) have extended chains at the C8-position of the DC-81 structure. During the synthesis of these compounds, a novel tin-mediated regiospecific cleavage reaction of the dioxole intermediate 18 was discovered, leading to the previously unknown iso-DC-81 (20). In addition, an unusual simultaneous nitration-oxidation reaction of 4-(3-hydroxypropoxy)-3-methoxybenzoic acid (8) was found to produce 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid (9), a key intermediate, in high yield. In general, the results of cytotoxicity and DNA-binding studies indicated that none of the changes made to the A-ring of the PBD system significantly improved either binding affinity or cytotoxicity in comparison to DC-81. This result suggests that the superior potency of natural products such as anthramycin (1), tomaymycin (2), and sibiromycin (3) is due entirely to differences in C-ring structure, and in particular exo or endo unsaturation at the C2-position and C2-substituents containing unsaturation. This study also provided information regarding the influence of A-ring substitution pattern on the relative stability of the interconvertible N10-C11 carbinolamine, carbinolamine methyl ether, and imine forms of PBDs.
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.
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.
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.
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.
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.
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.
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.
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.