ABSTRACT
The synthesis of a series of 35 substituted 3,4-diphenyl quinolines and isoquinolines is described. The majority of these molecules differ from all other triphenylethylene based antiestrogens by a different spatial location of the aminoalkyl side chain. The binding affinity of the most representative molecules (8, 9, 19, 20, 21, 23 and 25), including analogues 8 and 21 without the side chain, for the estrogen receptor alpha (ER) was determined. The ability of these molecules to induce the progesterone receptor was also studied. Antiproliferative activity was evaluated on MCF-7 human breast cancer cells, while intrinsic cytotoxic/cytostatic properties resulting from interaction with other targets than ER were assayed on L1210 murine leukemia cells. Introduction of an aminoalkylamino side chain at carbon 2 confers strong cytotoxic properties to diphenylquinolines 9 and 10 as well as pure antiestrogenic activities. However, cytotoxicity is so high with respect to antiestrogenicity that the latter was clearly observable only in one case (9b). The structure of compound 9b was determined by X-ray crystallography. Molecular modeling of its docking within the hormone-binding domain of the receptor was subsequently undertaken. According to our results, the design of molecules with the side chain bound to the ethylene part of the triphenyl ethylene skeleton might generate compounds of potential pharmacological interest.
Subject(s)
Drug Design , Estrogen Receptor Modulators/chemical synthesis , Estrogen Receptor Modulators/pharmacology , Isoquinolines/chemical synthesis , Isoquinolines/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Animals , Binding, Competitive/drug effects , Cell Division/drug effects , Crystallography, X-Ray , Estrogen Receptor Modulators/chemistry , Estrogen Receptor Modulators/metabolism , Humans , Isoquinolines/chemistry , Isoquinolines/metabolism , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Conformation , Molecular Structure , Quinolines/chemistry , Quinolines/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Tumor Cells, CulturedABSTRACT
Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthesized and evaluated for their anti-HIV-l activities. Key transformations include metalation at the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine (5) and its coupling with benzyl bromide or benzaldehyde derivatives. Biological studies revealed that some of the new 4-benzylpyridinones show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 14, 19, and 27, which inhibit the replication of HIV-1 in CEM-SS cells, with IC(50) values ranging from 0.2 to 6 nM are the most active compounds in this series. Biochemical studies showed that compound 27 strongly inhibited the activity of a recombinant HIV-1 RT. Moreover, the infectivity of isolated HIV-1 particles was severely decreased after exposure to compound 27. Although cross resistance is frequently observed between non-nucleoside reverse transcriptase inhibitors, compound 27 was capable of inhibiting a virus resistant to nevirapine with an IC(50) of 40 nM.
Subject(s)
Anti-HIV Agents/chemical synthesis , Pyridones/chemical synthesis , Reverse Transcriptase Inhibitors/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line , Cells, Cultured , Drug Resistance, Microbial , HIV-1/drug effects , Humans , Pyridones/chemistry , Pyridones/pharmacology , RNA-Directed DNA Polymerase/metabolism , Recombinant Proteins/antagonists & inhibitors , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Structure-Activity Relationship , Virion/drug effects , Virus Replication/drug effectsABSTRACT
Sequence-specific DNA recognition can be achieved by oligonucleotides that bind to the major groove of oligopyrimidine x oligopurine sequences. These intermolecular structures could be used to modulate gene expression and to create new tools for molecular biology. Here we report the synthesis and biochemical characterization of triple helix-specific DNA cleaving reagents. It is based on the previously reported triplex-specific ligands, benzo[e]pyridoindole (BePI) and benzo[g]pyridoindole (BgPI), covalently attached to ethylenediaminotetraacetic acid (EDTA). In the presence of iron, a reducing agent and molecular oxygen, BgPI-EDTA x FeII but not BePI-EDTA x FeII induced a double-stranded cut in a plasmid DNA at the single site where a triplex-forming oligonucleotide binds. At single nucleotide resolution, it was found that upon triplex formation BePI-EDTA x FeII led to cleavage of the pyrimidine strand and protection of the purine strand. BgPI-EDTA x FeII cleaved both strands with similar efficiency. The difference in cleavage efficiency between the two conjugates was rationalized by the location of the EDTA x FeII moiety with respect to the grooves of DNA (major groove: BePI-EDTA x FeII, minor groove: BgPI-EDTA x FeII). This work paves the way to the development of a new class of triple helix directed DNA cleaving reagents. Such molecules will be of interest for sequence-specific DNA cleavage and for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.
Subject(s)
DNA/chemistry , Edetic Acid/chemistry , Indoles/chemistry , Gene Expression Regulation/drug effects , Iron/chemistry , Models, Molecular , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Plasmids/chemistryABSTRACT
To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.
Subject(s)
Anti-HIV Agents/chemical synthesis , HIV Reverse Transcriptase/antagonists & inhibitors , Pyridones/chemical synthesis , Uracil/analogs & derivatives , Zalcitabine/chemistry , Zidovudine/chemistry , Anti-HIV Agents/pharmacology , Cytidine/analogs & derivatives , Cytidine/chemical synthesis , Cytidine/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , HIV-1/drug effects , HIV-2/drug effects , Molecular Structure , Pyridones/pharmacology , Structure-Activity Relationship , Uracil/chemical synthesis , Uracil/pharmacology , Zidovudine/analogs & derivatives , Zidovudine/chemical synthesis , Zidovudine/pharmacologyABSTRACT
BACKGROUND: Double-helical DNA can be recognized sequence specifically by oligonucleotides that bind in the major groove, forming a local triple helix. Triplex-forming oligonucleotides are new tools in molecular and cellular biology and their development as gene-targeting drugs is under intensive study. Intramolecular triple-helical structures (H-DNA) are expected to play an important role in the control of gene expression. There are currently no good probes available for investigating triple-helical structures. We previously reported that a pentacyclic benzoquinoquinoxaline derivative (BQQ) can strongly stabilize triple helices. RESULTS: We have designed and synthesized the first triple-helix-specific DNA cleaving reagent by covalently attaching BQQ to ethylenediaminetetraacetic acid (EDTA). The intercalative binding of BQQ should position EDTA in the minor groove of the triple helix. In the presence of Fe(2+) and a reducing agent, the BQQ-EDTA conjugate can selectively cleave an 80 base pair (bp) DNA fragment at the site where an oligonucleotide binds to form a local triple helix. The selectivity of the BQQ-EDTA conjugate for a triplex structure was sufficiently high to induce oligonucleotide-directed DNA cleavage at a single site on a 2718 bp plasmid DNA. CONCLUSIONS: This new class of structure-directed DNA cleaving reagents could be useful for cleaving DNA at specific sequences in the presence of a site-specific, triple-helix-forming oligonucleotide and also for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.
Subject(s)
DNA/chemistry , DNA/drug effects , Edetic Acid/chemical synthesis , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Quinoxalines/chemical synthesis , Base Pairing , Base Sequence , Drug Design , Edetic Acid/pharmacology , Hydrolysis , Models, Molecular , Molecular Sequence Data , Plasmids/drug effects , Quinoxalines/pharmacologyABSTRACT
Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.
Subject(s)
CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Purines/chemical synthesis , Purines/pharmacology , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle/drug effects , Cell Death/drug effects , Cell Survival/drug effects , Cell Survival/radiation effects , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 5 , Drug Evaluation, Preclinical , HeLa Cells/drug effects , HeLa Cells/radiation effects , Humans , Inhibitory Concentration 50 , Kinetin , Lethal Dose 50 , Nucleosides/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Purines/chemistry , S Phase/drug effects , Structure-Activity Relationship , Thymidine/pharmacokineticsABSTRACT
Analogues of the antitumor drug S 16020-2 modified at the 9, 10, or 11 position were synthesized and evaluated in vitro and in vivo on the P388 leukemia and B16 melanoma models. Starting from 9-methoxy-5, 11-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylic acid ethyl ester, the 11-CH3 analogue of 9-hydroxy-5,6-dimethyl-6H-pyrido[4, 3-b]carbazole-1-carboxylic (2-(dimethylamino)ethyl)amide (1), compound 4, was synthesized using a four-step sequence, whereas its 10-CH3 analogue 5 was prepared using a two-step pathway, starting from compound 1. Finally starting from the 9-OH compounds 1, 4, and 5, a series of variously 9-O-substituted derivatives were synthesized. In these series, the most active compounds resulted from esterification of the 9-OH group with various aliphatic diacids, which led to 9-O-CO-( )-COOH derivatives of 1, 4, and 5. For these compounds, the number of long-term surviving mice obtained at the optimal dose were 60-100% in the ip/iv P388 leukemia and 10-35% in the ip/ip B16 melanoma, corresponding to an improved therapeutic index with respect to 1 and 4. This high antitumor activity, with curative examples in both models, was not due to a higher cytotoxicity since these compounds were equally or slightly less potent in vitro than 1 and 4. The most active compounds were thus selected for further in vivo evaluation.
Subject(s)
Antineoplastic Agents/chemical synthesis , Ellipticines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Drug Screening Assays, Antitumor , Ellipticines/chemistry , Ellipticines/pharmacology , Inhibitory Concentration 50 , Leukemia P388/drug therapy , Leukemia P388/pathology , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Methylation , Mice , Neoplasm Transplantation , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Oligonucleotide-directed triple helix formation is mostly restricted to oligopyrimidine*oligopurine sequences of double helical DNA. An interruption of one or two pyrimidines in the oligopurine target strand leads to a strong triplex destabilisation. We have investigated the effect of nucleotide analogues introduced in the third strand at the site opposite the base pair inversion(s). We show that a 3-nitropyrrole derivative (M) discriminates G*C from C*G, A*T and T*A in the presence of a triplex-specific ligand (a benzo[e]pyridoindole derivative, BePI). N6-methoxy-2,6-diaminopurine (K) binds to an A*T base pair better than a T*A, G*C or C*G base pair. Some discrimination is still observed in the presence of BePI and triplex stability is markedly increased. These findings should help in designing BePI-oligonucleotide conjugates to extend the range of DNA sequences available for triplex formation.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Chromosome Inversion , DNA/drug effects , Hydrogen Bonding , Indoles/pharmacology , Ligands , Oligonucleotides , Purines , Pyridines/pharmacology , PyrimidinesABSTRACT
The title compounds were synthesized in 9-10 steps in order to compare their cytotoxic properties to that for 1-(3-dimethylaminopropyl)-amino-4,5-dimethyl- 8-hydroxy-5H-pyrido[4,3-b]indole. Whereas the latter is a potent cytotoxic agent, displaying significant antitumour activity, the corresponding 9-propyl (and 7,9-dimethyl) derivatives were found to be > 10-fold less cytotoxic.
Subject(s)
Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Indoles/pharmacology , Mice , Structure-Activity Relationship , Tumor Cells, Cultured/drug effectsABSTRACT
Furo[3,2-e]- and pyrano[3,2-e]pyrido[4,3-b] indoles were synthesized from 1,4,5-trisubstituted 8-hydroxy-5H-pyrido[4,3-b]indoles. The intermediates, 10-chloro-6H-furo[3,2-e]pyrido[4,3-b]indole (11), 10-chloro-2,6-dihydro-1H-furo[3,2-e]pyrido-[4,3-b]indole (10) and 11-chloro-2,3-dihydro-3H,7H-pyrano[3,2-e]pyrido[4,3-b]indole (15), were substituted by diamines under thermal conditions (180 degrees C). In contrast, 11-chloro-3H,7H-pyrano[3,2-e]pyrido[4,3-b]indole (14), 9-allyl-1-chloro-4,5-dimethyl-5H-pyrido[4,3-b]indole (9a) and 8-propargyloxy-4,5-dimethyl-5H-pyrido[4,3-b]indole (8) led mainly to 1-aminosubstituted 8-hydroxy-5H-pyrido[4,3-b]indole derivatives resulting from an unexpected C3 unit elimination. When examined in three tumour cell lines (L1210 leukaemia, the B16 melanoma and the MCF7 breast adenocarcinoma) the new amino substituted furo[3,2-e]-, dihydrofuro[3,2-e]- and dihydropyrano[3,2-e]-pyrido[4,3-b]indole derivatives revealed cytotoxic properties, especially important for the 2,6-dihydro-1H-furo[3,2-e]pyrido[4,3-b]indole series. The most active compound (12b) significantly inhibits both DNA topoisomerases I and II, and is as potent as Adriamycin at inhibiting cell proliferation and inducing a massive accumulation of L1210 cells in the G2 + M phase of the cell cycle. However, 12b was less active than Adriamycin when tested in vivo against P388 leukaemia or the B16 melanoma tumour models.
Subject(s)
Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Humans , Indoles/pharmacology , Indoles/therapeutic use , Isomerases/antagonists & inhibitors , Mice , Neoplasms, Experimental/drug therapy , Tumor Cells, Cultured/drug effectsABSTRACT
DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3, 4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.
Subject(s)
DNA/chemistry , Models, Molecular , Nucleic Acid Conformation , Quinoxalines/chemistry , Animals , DNA/genetics , Gene Expression Regulation , Humans , LigandsABSTRACT
The synthesis of a new family of inhibitors of the cell cycle regulating cyclin-dependent kinases (CDK's) is reported. These compounds, related to the purines olomoucine and roscovitine, are characterised by the presence of alkynylated side chains at C2. They inhibit CDK's with IC50's in the 200 nM range.
Subject(s)
Alkynes/chemical synthesis , CDC2 Protein Kinase/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Purines/chemical synthesis , Alkynes/chemistry , Alkynes/pharmacology , Animals , CDC2 Protein Kinase/isolation & purification , Cyclin B/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , Kinetin , Oocytes/enzymology , Purines/chemistry , Purines/pharmacology , Roscovitine , Starfish , Structure-Activity RelationshipABSTRACT
The synthesis of a DNA-cutting agent that conjugates an ellipticine chromophore and a copper complex of bis(salicylidene)ethylenediamine, referred to as a salen, is reported. The presence of the salen.Cu complex allows cleavage of DNA via oxygen-based radicals, and the ellipticine moiety serves as a DNA anchor. Spectroscopic measurements indicate that the intercalation geometry of the ellipticine chromophore is preserved with the hybrid. The cleavage is much more efficient with the conjugate than with the Schiff base copper complex alone.
Subject(s)
Chelating Agents/chemistry , Chelating Agents/metabolism , Copper/chemistry , Copper/metabolism , DNA Damage , DNA/drug effects , DNA/metabolism , Ellipticines/chemical synthesis , Ellipticines/metabolism , Ethylenediamines/chemistry , Ethylenediamines/metabolism , Organometallic Compounds/chemical synthesis , Organometallic Compounds/metabolism , Chelating Agents/pharmacology , Circular Dichroism , Copper/pharmacology , Ellipticines/pharmacology , Ethylenediamines/pharmacology , Organometallic Compounds/pharmacology , SpectrophotometryABSTRACT
Two genistein analogues (MD831 and MD833) have been synthesized and analyzed for their biological properties and their mechanism of action im comparison to genistein either in vitro or in intact cells. We showed that, in vitro, one of these compounds (MD831) inhibits the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR) as efficiently as genistein. However, treatment of A431 cells with these compounds did not result in any significant modification of EGFR tyrosine phosphorylation. Extracellular-signal regulated kinase (ERK) phosphorylation in cells stimulated by EGF was enhanced in the presence of MD831, whereas the other compounds, genistein and MD833, were able to activate the c-jun N-terminal kinase (JNK). This study showed that two structurally related compounds could elicit markedly different pharmacological effects on two signalling pathways, one involved in the mitogenic response and the other in the stress response. Such compounds may be useful to characterize signalling events involved in cell response to physiological stimuli.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , ErbB Receptors/drug effects , Isoflavones/pharmacology , Mitogen-Activated Protein Kinases , Signal Transduction/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/drug effects , ErbB Receptors/metabolism , Genistein , In Vitro Techniques , Isoflavones/chemical synthesis , Isoflavones/chemistry , JNK Mitogen-Activated Protein Kinases , Phosphorylation/drug effects , Tumor Cells, CulturedABSTRACT
The stabilization of triple-helical DNA under physiological conditions is an important goal for the control of gene expression using the antigen strategy, an approach whereby an oligonucleotide binds to the major groove of double-helical DNA to fom a triple helix. To this end, triplex-specific intercalators, namely benzopyridoindole (BPI) and benzopyridoquinoxaline (BPQ) derivatives, have been conjugated to the 5' end or to an internucleotide position of a 15-mer oligonucleotide. These conjugates were then tested, using thermal denaturation experiments, for their ability to form and stabilize a triple-helical structure involving a 42-mer duplex target. All of the conjugates were found to do so. The B[h]PQ derivatives stabilized particularly well when attached to the 5' end with a delta Tm of 15 degrees C and -delta delta G degrees 37 of 3.4 kcal mol-1 (pH 6.9, 140 mM KCI, 15 mM sodium cacodylate, 2 mM MgCl2, 0.8 mM spermine). Though most of the derivatives when attached to the internucleotide position were not able to stabilize triple-helical DNA as well as when attached to the 5' end, one B[f]PQ derivative with an internucleotide attachment did so, with a delta Tm of 13 degrees C and -delta delta G degrees 37 of 2.8 kcal mol-1. To a lesser degree, these conjugates were also able to stabilize duplex structures with single-stranded targets. Results were compared to the stabilization obtained with acridine conjugates as well as to a similar study performed with a different sequence.
Subject(s)
DNA/metabolism , Intercalating Agents/metabolism , Interleukin-2/chemistry , Oligonucleotides/metabolism , Promoter Regions, Genetic , Hydrogen-Ion Concentration , Nucleic Acid Conformation , ThermodynamicsABSTRACT
The antitumor drug pazelliptine (PZE) binds to natural and synthetic DNA sequences at 100 mM NaCl, pH 7.0, as deduced from the absorption and fluorescence data. Scatchard plots constructed from the results obtained with poly(dG-dC)-poly(dG-dC) give binding constants of base pairs in the range (2-6) x 10(5)M(-1). The modifications in the absorption and fluorescence spectra observed when PZE binds to various polynucleotides, namely poly(dA-dT)-poly(dA-dT), poly(dA)-poly(dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA, reveal a change in the protonation state of the drug upon binding, increasing the apparent pKa of its 9-N-nitrogen atom. The PZE excited state properties serve as a sensitive probe to distinguish between homo and hetero A-T sites as well as between AT and GC sites. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to the bound PZE chromophore, a result consistent with an intercalative mode of binding of the drug to DNA. The emission is enhanced when PZE is bound to A-T base pairs (approximately 30% increase of phi(F) whereas it is quenched in the vicinity of G-C base pairs (approximately 90% decrease of phi(F)). Furthermore, the fluorescence spectrum obtained with calf thymus DNA is hardly distinguishable from that obtained with poly(dG-dC)-poly(dG-dC), suggesting a binding of PZE to G-C rich regions.
Subject(s)
Antineoplastic Agents/pharmacology , DNA/drug effects , Indoles/pharmacology , Isoquinolines/pharmacology , DNA/chemical synthesis , Energy Transfer , Models, Chemical , Polyribonucleotides , Spectrum AnalysisABSTRACT
Starting from 2-(6-methoxy-1-methylcarbazol-2-yl)ethylamine and diethyl-2,6-pyridine dicarboxylate, the title compounds were obtained through five or six steps. The new compounds retained significant cytotoxicity towards various tumor cell lines, but in vivo studies on murine P388 leukemia, B16 melanoma and Lewis lung carcinoma showed a lowered antitumor activity with respect to that of the related olivacine lead compound 1.
Subject(s)
Antineoplastic Agents/chemical synthesis , Pyridines/chemical synthesis , Animals , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Lewis Lung/drug therapy , Cell Survival/drug effects , DNA, Neoplasm/drug effects , Doxorubicin/pharmacology , Drug Screening Assays, Antitumor , Humans , Leukemia P388/drug therapy , Melanoma, Experimental/drug therapy , Mice , Pyridines/pharmacology , Tumor Cells, CulturedABSTRACT
We have examined the effects of benzopyridoindole derivatives on triple helices with antiparallel third strands. Absorption spectroscopy, footprinting, and gel retardation experiments demonstrate that a benzopyridoindole derivative (BePI) is able to induce formation of a triple helix with an antiparallel (G, T)-containing third strand, which does not form in the absence of this ligand. This triple-helical complex is very stable with a half-dissociation temperature as high as 51 degrees C, and its formation is pH independent. Antiparallel oligonucleotides containing thymine and guanine bind strongly to double-helical DNA under physiological conditions in the presence of only 0.5 microM BePI. Formation of a BePI-stabilized triple helix strongly inhibits cleavage of the target duplex by DNase I.
Subject(s)
DNA/chemistry , DNA/chemical synthesis , Nucleic Acid Conformation , Base Sequence , DNA/isolation & purification , DNA Footprinting , Drug Stability , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Indicators and Reagents , Indoles , Ligands , Molecular Sequence Data , Pyridines , Spectrophotometry, UltravioletABSTRACT
Biophysical, footprinting, and chemical probing experiments are described which characterize the triple helix-stabilizing effects of a benzo[f]pyridoquinoxaline derivative BfPQ-4,3 structurally related to the previously reported benzo[f]pyridoindole compound BePI [Mergny et al. (1992) Science 256, 1681-1684]. Two parallel triple helix model systems have been investigated; one in which the third strand matched perfectly a 27 base pair purine-pyrimidine motif in target DNA and another in which the third strand was one nucleotide longer, i.e., a 28-mer. In the latter system, the pairing of the (Y)28 third strand to the (Y.R)27 target induces the formation of a bulge containing at least one unpaired base, which can be evidenced by chemical probing experiments with osmium tetroxide. BPQ, which uinwinds a duplex DNA by 17 degrees as judged by viscometric experiments and otherwise behaves as a typical nonspecific intercalculating drug, promotes the formation of Y.R.Y parallel triple helix containing both T.A.T and C.G.C+ triplets. Both DNase I and MPE.FeII footprinting experiments concur that triplex formation with the target (Y.R)27 sequence can be detected in the presence of BPQ at about 10-fold lower oligonucleotide concentrations than are required to produce an equivalent footprint in the absence of the drug. In addition, BPQ will promote binding to the polypurine-polypyrimidine target sequence by the longer mismatched oligonucleotide, providing significant stabilization of the parallel bulge-containing(Y.R)27,(Y)28 triplex with nearly the same efficiency as the bulge-free (Y.R)27.(Y)28 triplex. Thus in vivo BPQ might enhance the formation of both undesired and desired DNA triplexes. By performing an MPE*FeII probing reaction with a 5'-32 P-labeled oligonucleotide third strand, we have obtained evidence that BPQ is actually bound to the triplex region and may distort in a sequence-specific fashion.