ABSTRACT
Bisbenzimidazoles with terminal alkynyl linkers, selective inhibitors of bacterial topoisomerase I, have been evaluated using bacterial cytological profiling (BCP) to ascertain their mechanism of action and screened for synergism to improve Gram-negative bacterial coverage. Principal component analysis of high throughput fluorescence images suggests a dual-mechanism of action affecting DNA synthesis and cell membrane integrity. Fluorescence microscopy of bacteria challenged with two of the alkynyl-benzimidazoles revealed changes in the cellular ultrastructure that differed from topoisomerase II inhibitors including induction of spheroplasts and membrane lysis. The cytoskeleton recruitment enzyme inhibitor A22 in combination with one of the alkynyl-benzimidazoles was synergistic against Acinetobacter baumannii and Escherichia coli. Gram-positive coverage remained unchanged in the A22-alkynyl bisbenzimidazole combination. Efflux inhibitors were not synergistic, suggesting that the Gram-negative outer membrane was a significant barrier for alkynyl-bisbenzimidazole uptake. Time-kill assays demonstrated the A22-bisbenzimidazole combination had a similar growth inhibition curve to that of norfloxacin in E.coli. Bisbenzimidazoles with terminal alkynyl linkers likely impede bacterial growth by compromising cell membrane integrity and by interfering with DNA synthesis against Gram-positive pathogens and in the synergistic combination against Gram-negative pathogens including E. coli and multidrug-resistant A. baumanii.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Bisbenzimidazole/analogs & derivatives , Escherichia coli/drug effects , Topoisomerase I Inhibitors/pharmacology , Anti-Bacterial Agents/chemistry , Bisbenzimidazole/pharmacology , Cell Membrane/drug effects , Drug Synergism , Topoisomerase I Inhibitors/chemistryABSTRACT
The vacuolar (Hâº)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I-IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a-e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 µM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.
Subject(s)
Antineoplastic Agents/chemistry , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemistry , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Bisbenzimidazole/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor/methods , Female , Humans , Ovarian Neoplasms/drug therapy , Triple Negative Breast Neoplasms/drug therapyABSTRACT
A bisoxyphenylene-bisbenzimidazole series with increasing aliphatic chain length (CH2 to C10 H20 ) containing a meta- (m) or para (p)-benzimidazole linkage to the phenylene ring was tested for ability to inhibit the growth of metronidazole-susceptible (C1) and metronidazole-refractory (085) Trichomonas vaginalis isolates under aerobic and anaerobic conditions. Compound 3m, 2,2'-[α,ω-propanediylbis(oxy-1,3-phenylene)]bis-1H-benzimidazole, displayed a 5.5-fold lower minimum inhibitory concentration (MIC) toward T. vaginalis isolate 085 than metronidazole under aerobic growth conditions, (26 µm compared to 145 µm). A dose of 25 mg/kg per day for four days of compound 3m cured a subcutaneous mouse model infection using T. vaginalis isolates 286 (metronidazole susceptible) and 085 (metronidazole refractory). Compound 3m was weakly reduced by pyruvate:ferredoxin oxidoreductase, but unlike metronidazole was not dependent upon added ferredoxin. It is concluded from structure-activity relationships that there was no obvious trend based on the length of the central aliphatic chain, or the steric position of the bisbenzimidazole enabling prediction of biological activity. The compounds generally fulfill Lipinski's rile of five, indicating their potential as drug leads.
Subject(s)
Antiprotozoal Agents/chemistry , Antiprotozoal Agents/therapeutic use , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/therapeutic use , Drug Resistance , Trichomonas Vaginitis/drug therapy , Trichomonas vaginalis/drug effects , Animals , Antiprotozoal Agents/pharmacology , Bisbenzimidazole/pharmacology , Cell Line, Tumor , Female , Humans , Metronidazole/pharmacology , Mice , Microbial Sensitivity Tests , Trichomonas vaginalis/growth & developmentABSTRACT
A series of new fluorescent symmetric dimeric bisbenzimidazoles DBP(n) bearing bisbenzimidazole fragments joined by oligomethylene linkers with a central 1,4-piperazine residue were synthesized. The complex formation of DBP(n) in the DNA minor groove was demonstrated. The DBP(n) at micromolar concentrations inhibit in vitro eukaryotic DNA topoisomerase I and prokaryotic DNA methyltransferase (MTase) M.SssI. The DBP(n) were soluble well in aqueous solutions and could penetrate cell and nuclear membranes and stain DNA in live cells. The DBP(n) displayed a moderate effect on the reactivation of gene expression.
Subject(s)
Bisbenzimidazole/analogs & derivatives , DNA/chemistry , DNA/drug effects , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Bisbenzimidazole/chemical synthesis , Bisbenzimidazole/pharmacology , Cell Line , DNA/genetics , DNA-Cytosine Methylases/antagonists & inhibitors , Dimerization , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/chemistry , Gene Expression/drug effects , Humans , MCF-7 Cells , Mice , Microscopy, Fluorescence , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/pharmacologyABSTRACT
3,5-Bis(trifluoromethyl)benzene modified bisbenzimide H 33258 was synthesized as a (19)F magnetic resonance-based DNA detection probe. The chemical shift and fluorescence of the probe were significantly changed by the addition of hairpin DNAs having an AATT sequence. The probe enables (19)F NMR/fluorescence bimodal detection of the model DNA double strands simultaneously.
Subject(s)
Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemistry , DNA Probes/chemistry , DNA/analysis , Fluorescence , Toluene/analogs & derivatives , Bisbenzimidazole/chemical synthesis , DNA Probes/chemical synthesis , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Microscopy, Fluorescence , Molecular Structure , Toluene/chemistryABSTRACT
In this study, a new series of bis-benzimidazole derivatives were designed and synthesized. Most of these new compounds showed significant anti-tumor activity in vitro compared to Hoechst 33258. Among them, the most potent compound 8 had the IC(50) values of 0.56µM for HL60 (Human promyelocytic leukemia cells) tumor cell line and 0.58µM for U937 (Human leukemic monocyte lymphoma cells) tumor cell line. Subsequent toxicity study on human peripheral blood mononuclear cells (PBMC) showed that compound 8 exhibited less toxicity than 5-FU. We also found that apoptosis and autophagy were simultaneously induced by compound 8 in HL60 cells, and inhibition of autophagy by 3-MA decreased compound 8-induced apoptosis, indicating that they acted in synergy to exert tumor cell death.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/pharmacology , Antineoplastic Agents/chemistry , Bisbenzimidazole/chemical synthesis , Bisbenzimidazole/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HL-60 Cells , HeLa Cells , Humans , Leukocytes, Mononuclear/drug effects , Molecular Structure , Structure-Activity Relationship , U937 CellsABSTRACT
Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.
Subject(s)
Drug Design , Oligodeoxyribonucleotides/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antiviral Agents/chemistry , Base Sequence , Benzoxazoles/chemistry , Berberine/chemistry , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemistry , Butyrates , Hydrogen Bonding , Imidazoles/chemistry , Indoleacetic Acids/chemistry , Ligands , Molecular Dynamics Simulation , Netropsin/analogs & derivatives , Netropsin/chemistry , Nucleic Acid Conformation , Plasmids/chemistry , Vincristine/chemistryABSTRACT
Double-stranded DNA is a one of the most important intracellular anticancer agent targets. Disturbance of DNA functions as well as DNA structure lead to disorder of such processes as transcription and/or translation thus inducing tumor cells death. Complex formation between novel dimeric bisbenzimidazole DB(7) and poly(dA-dT) duplex in comparison with known monomeric bisbenzimidazole MB(Ac) was investigated in this study. DB(7)-poly(dA-dT) binding constant was determined by fluorescence spectroscopy using Scatchard plot and it values 1.18 x 10(8) M(-1) that is two orders of magnitude larger than MB(Ac) one (2.06 x 10(6) M(-1)). Thus, from findings mentioned above it could be concluded that the presence of two bisbenzimidazole moieties in the ligand structure significantly increases its affinity to the polynucleotide which motivates the synthesis of new potential anticancer drugs based on dimeric bisbenzimidazoles.
Subject(s)
Antineoplastic Agents/chemistry , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemistry , DNA/chemistry , Fluorescent Dyes/chemistry , Oligodeoxyribonucleotides/chemistry , Antineoplastic Agents/pharmacology , Bisbenzimidazole/pharmacology , Cell Death/drug effects , Fluorescent Dyes/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolismABSTRACT
Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV(A)Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Photosensitivity Disorders , Bisbenzimidazole/analogs & derivatives , DNA/chemistry , DNA/drug effects , DNA/radiation effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , K562 Cells , LigandsABSTRACT
HIV-1 integrase is responsible for one of the key steps of the viral replication, integration of the viral cDNA into the host cell genome. Integration inhibition leads to complete block of the virus replication. In this study inhibition of integration by dimeric bisbenzimidazoles DBBI(7) with heptamethylene and DBBI(8) with tri(ethylene glycol) spacers was examined, and it was learned out that IC50 for DBBI(7) was about 0.03 microM, and IC50 for DBBI(8) was about 10 microM. By using cross-linking assays, it was shown that both compounds impeded a proper disposition of DNA-substrate at the active centre of integrase. Dissociation constants for complexes between either DBBI and DNA-substrate of integrase were determined. Calculated Kd values were 270 nM and 140 nM for complexes formed by DBBI(7) and DBBI(8), respectively. Therefore, inhibition of integration does not directly result from the binding of DBBIs with DNA. The dependence of initial rates of enzymatic reaction on the DNA-substrate concentration in presence of different concentrations of inhibitors was found, and inhibition constants were determined. All the data obtained allow us to suppose that the different inhibition activity of DBBI(7) and DBBI(8) results from the different mechanism of their binding: DBBI(7) is a competitive inhibitor of integrase whereas DBBI(8) is assumed to show a more complex mechanism of inhibition.
Subject(s)
Bisbenzimidazole/chemistry , DNA/chemistry , HIV Integrase Inhibitors/chemistry , HIV Integrase/chemistry , HIV-1/enzymology , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/metabolism , Catalytic Domain , DNA/metabolism , HIV Integrase/metabolism , Kinetics , Protein BindingABSTRACT
Herein, we describe the design of high affinity ligands that bind expanded rCUG and rCAG repeat RNAs expressed in myotonic dystrophy type 1 (DM1) and spinocerebellar ataxia type 3. These ligands also inhibit, with nanomolar IC(50) values, the formation of RNA-protein complexes that are implicated in both disorders. The expanded rCUG and rCAG repeats form stable RNA hairpins with regularly repeating internal loops in the stem and have deleterious effects on cell function. The ligands that bind the repeats display a derivative of the bisbenzimidazole Hoechst 33258, which was identified by searching known RNA-ligand interactions for ligands that bind the internal loop displayed in these hairpins. A series of 13 modularly assembled ligands with defined valencies and distances between ligand modules was synthesized to target multiple motifs in these RNAs simultaneously. The most avid binder, a pentamer, binds the rCUG repeat hairpin with a K(d) of 13 nM. When compared to a series of related RNAs, the pentamer binds to rCUG repeats with 4.4- to >200-fold specificity. Furthermore, the affinity of binding to rCUG repeats shows incremental gains with increasing valency, while the background binding to genomic DNA is correspondingly reduced. Then, it was determined whether the modularly assembled ligands inhibit the recognition of RNA repeats by Muscleblind-like 1 (MBNL1) protein, the expanded-rCUG binding protein whose sequestration leads to splicing defects in DM1. Among several compounds with nanomolar IC(50) values, the most potent inhibitor is the pentamer, which also inhibits the formation of rCAG repeat-MBNL1 complexes. Comparison of the binding data for the designed synthetic ligands and MBNL1 to repeating RNAs shows that the synthetic ligand is 23-fold higher affinity and more specific to DM1 RNAs than MBNL1. Further studies show that the designed ligands are cell permeable to mouse myoblasts. Thus, cell permeable ligands that bind repetitive RNAs have been designed that exhibit higher affinity and specificity for binding RNA than natural proteins. These studies suggest a general approach to targeting RNA, including those that cause RNA dominant disease.
Subject(s)
Drug Design , Machado-Joseph Disease/drug therapy , Machado-Joseph Disease/genetics , Myotonic Dystrophy/drug therapy , Myotonic Dystrophy/genetics , RNA, Messenger/metabolism , Trinucleotide Repeat Expansion/genetics , Animals , Base Sequence , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/metabolism , Bisbenzimidazole/pharmacology , Bisbenzimidazole/therapeutic use , Cell Line , Humans , Inhibitory Concentration 50 , Inverted Repeat Sequences/genetics , Ligands , Mice , Permeability , Protein Binding/drug effects , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Reproducibility of Results , Substrate SpecificityABSTRACT
PURPOSE: The aim of the study is to establish the relationship between the efficiency of DNA double-stranded breakage by (125)I-labelled DNA ligands and the distance from the decaying atom to the helical axis. MATERIALS AND METHODS: Two new iodinated minor groove binding ligands were synthesized which, on the basis of molecular modelling studies, place the iodine atom at different distances from the DNA helical axis (namely 7.4 and 11.2 A degrees ). Plasmid DNA breakage experiments, in both buffer-only and buffer + 2M dimethylsulfoxide (DMSO), were used to determine the efficiency of induction of internal double-stranded breaks (DSB) of the two new ligands, as well as that for (125)I-Hoechst 33258, which is characterized by a helical axis-iodine atom distance of 9.1 A degrees . RESULTS: The results showed a progressive decrease in the efficiency of DNA DSB induction with the axis-iodine atom distance, for both incubation conditions. The distance-damage relationship was somewhat steeper than previously predicted from the theoretical studies by Humm and Charlton, based on radical-mediated damage. Another distinctive trend was revealed by comparison of breakage efficiency with and without DMSO. The extent of DMSO protection increased significantly with DNA-iodine distance. CONCLUSIONS: The steeper than predicted decrease in DSB induction with DNA-iodine distance is consistent with a substantial contribution to DNA breakage of the charge neutralization effect (arising from the transient positive charge left on the daughter Te atom), and the expectation that this contribution would be very dependent on the distance of the site of hole injection from the base-pair pi-stack. An important caveat to the results and conclusions is the need to confirm the estimated helical axis-iodine distances with X-ray crystallography studies, and for further exemplification with a more extensive collection of DNA ligands.
Subject(s)
Benzimidazoles/chemistry , Bisbenzimidazole/analogs & derivatives , DNA Breaks, Double-Stranded/radiation effects , Iodine Radioisotopes/chemistry , Bisbenzimidazole/chemistry , Ligands , Models, Molecular , Nucleic Acid Conformation , Plasmids/chemistry , Plasmids/radiation effects , RadioactivityABSTRACT
We present a strategy to generate a light-up fluorophore-aptamer pair. The strategy was based on a modification of a conventional DNA-staining dye to suppress its affinity to the original targets, and subsequent re-selection of aptamers that would bind to the modified dye. Along this line, we prepared an environmental polarity-sensitive Hoechst derivative with low affinity to the usual AT-rich dsDNA targets. DNA aptamers, in vitro selected from a random pool, worked as a trigger to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative.
Subject(s)
Aptamers, Nucleotide/chemistry , Bisbenzimidazole/analogs & derivatives , Fluorescent Dyes/chemistry , DNA/chemistry , DNA Probes/chemistryABSTRACT
An equilibrium containing the thiol derivative of Hoechst33258 (Ht-SH), glutathione (G-SH), and the corresponding homo and hetero disulfides was shifted by the addition of the duplex DNA. It was shown from the analysis of the components that the hetero disulfide Ht-SS-G increased by binding with the DNA (CA14) with an A(3)T(3) binding motif for the structure of Hoechst33258, and that the different equilibrium shift was observed in the presence of CT14 with no A(3)T(3) binding motif.
Subject(s)
Bisbenzimidazole/analogs & derivatives , DNA/chemistry , Disulfides/chemistry , Glutathione/chemistry , Base Sequence , Ligands , Oligonucleotides/chemistry , Oxidation-ReductionABSTRACT
Tethering of BBZPNH2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine-polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 degrees C and double helix to single strand transition by 16 degrees C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.
Subject(s)
Benzimidazoles/chemistry , Bisbenzimidazole/chemistry , Bisbenzimidazole/chemical synthesis , DNA/chemistry , Maleimides/chemistry , Sulfhydryl Compounds/chemistry , Benzimidazoles/chemical synthesis , Bisbenzimidazole/analogs & derivatives , Circular Dichroism , DNA/chemical synthesis , Molecular Structure , Nucleic Acid Conformation , Oligonucleotides/chemistry , Structure-Activity Relationship , TemperatureABSTRACT
The Auger electron-emitting isotope 123I is of interest in the context of potential exploitation of Auger electron emitters in radioimmunotherapy. The efficiency of induction of cytotoxic lesions by decay of DNA-associated 125I, the prototype Auger electron emitter, is well established, but its long half-life (60 days) is a limitation. However, the advantage of the much shorter half-life of 123I (13.2 h) might be outweighed by its "weaker" Auger electron cascade with an average of 8-11 Auger electrons, compared to about 15-21 electrons for 125I. Accordingly, the efficiency of DNA breakage for DNA-associated 123I was investigated by incubation of 123I-iodoHoechst 33258 with plasmid DNA. The efficiency of double-strand break induction by decay of 123I was 0.62 compared to 0.82 per decay of 125I in the same experimental system. In the presence of dimethylsulfoxide, the values were 0.54 and 0.65 for decay of 123I and 125I, respectively. The results also showed that at a very low ligand/plasmid molar ratio (<1), the majority of cleavage seemed to occur at a particular site on the plasmid molecule, indicating preferential binding of the 123I-ligand to a unique site or a cluster of neighboring sites.
Subject(s)
DNA Damage/radiation effects , DNA/metabolism , Electrons , Plasmids/metabolism , Bisbenzimidazole/analogs & derivatives , Half-Life , Iodine Radioisotopes , LigandsABSTRACT
Chiral hairpin polyamides linked to a Hoechst 33258 analogue at the alpha-position of the hairpin turn amino acid (1,2) were synthesized on solid phase by adopting Fmoc and ivDde techniques. The DNA-binding properties of enantiomeric conjugates 1 and 2, and N-terminal linked conjugate 3 for 8-14bp sequences were determined by spectrofluorometric and thermal melting studies. Conjugates 1 and 2 recognize a 10bp sequence, while conjugate 3 recognizes a 9bp sequence. Interestingly, R-enantiomer 1 exhibited 10- to 30-fold higher binding affinities than S-enantiomer 2 for the DNA sequences studied. These binding differences were accounted for by molecular modeling studies, which revealed that the amide proton nearest to the chiral center in R-conjugate 1 is better positioned to form hydrogen bonds to the DNA bases, while S-conjugate 2 does not.
Subject(s)
Bisbenzimidazole/analogs & derivatives , DNA/chemistry , Nylons/chemistry , Pyrroles/chemistry , Base Pairing , Base Sequence , Binding Sites , Kinetics , Models, Molecular , Nucleic Acid Conformation , Structure-Activity Relationship , ThermodynamicsABSTRACT
A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.
Subject(s)
Bisbenzimidazole/toxicity , DNA Topoisomerases/metabolism , DNA/metabolism , Nitrosourea Compounds/toxicity , Topoisomerase Inhibitors , Animals , Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemical synthesis , Cattle , Cell Line, Tumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/toxicity , Humans , Nitrosourea Compounds/chemical synthesis , Protein Binding/drug effects , Protein Binding/physiologyABSTRACT
The factors responsible for the binding of Hoechst 33258 with DNA residues have been investigated in this work using the AM1 method. First and foremost, it is found that, although all crystal structure determinations indicate a preference for binding at AT rich sites, the hydrogen bond strength is actually greater for complexes with cytosine and guanine. From this, it has been inferred that other factors such as electrostatic, van der Waals interactions and nonbonded contacts with the walls of the minor groove have a strong role to play in the binding process. The hydrogen bond is found to be stronger for complexation with the thymine O2 than with the adenine N3, in line with experimental observations. Combined QM/MM studies on the drug complexed with the Dickerson-Drew dodecamer reveal that binding induces structural changes in both the ligand as well as DNA. Electron donating substituents at the para position in the phenyl ring of Hoechst 33258 lead to stronger binding with DNA. A correlation with the octanol/water partition coefficients points to the importance of hydrophobic and electrostatic interactions.
Subject(s)
Bisbenzimidazole/analogs & derivatives , Bisbenzimidazole/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Adenine/chemistry , Benzimidazoles/chemistry , Crystallography, X-Ray , Cytosine/chemistry , DNA/chemistry , Guanine/chemistry , Hydrogen/chemistry , Hydrogen Bonding , Ligands , Macromolecular Substances/chemistry , Models, Chemical , Nucleic Acid Conformation , Octanols/chemistry , Protein Binding , Protein Conformation , Spectrophotometry , Static Electricity , Thermodynamics , Thymine/chemistry , Water/chemistryABSTRACT
(2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide (compound E) is a gamma-secretase inhibitor capable of reducing amyloid beta-peptide (1-40) and amyloid beta-peptide (1-42) levels. In this study we investigated the effect of in vivo administration of compound E on guinea-pig plasma, CSF and cortical amyloid beta-peptide (1-40) concentration. Using repeated sampling of CSF, compound E (30 mg/kg p.o.) was shown to cause a time-dependent decrease in CSF amyloid beta-peptide (1-40) levels, which was maximal at 3 h (70% inhibition), compared to baseline controls. After 3 h administration, compound E (3, 10 and 30 mg/kg p.o.), reduced plasma, CSF and DEA-extracted cortical amyloid beta-peptide (1-40) levels by 95, 97 and 99%; 26, 48 and 78%; 32, 33, and 47%, respectively, compared to vehicle control values. In the same animals, compound E (3, 10 and 30 mg/kg p.o.) inhibited cortical gamma-secretase activity, determined ex vivo using the recombinant substrate C100Flag, by 40, 71 and 79% of controls, respectively. These data demonstrate the value of determining not only the extent by which systemic administration of a gamma-secretase inhibitor reduces amyloid beta-peptide, but also the inhibition of brain gamma-secretase activity, as a more direct estimate of enzyme occupancy.