RESUMO
A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.
Assuntos
Bisbenzimidazol/química , Bisbenzimidazol/farmacologia , DNA/química , Aliivibrio/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sequência de Bases , DNA/genética , Desenho de Fármacos , Escherichia coli/metabolismo , Corantes Fluorescentes , Integrase de HIV , Inibidores de Integrase de HIV/farmacologia , Ligantes , Estrutura Molecular , Pirróis , Percepção de Quorum/fisiologia , Relação Estrutura-AtividadeRESUMO
A series of DNA minor groove binding fluorescent dimeric bisbenzimidazoles DBA(n) bearing linkers of various length were synthesized and their biochemical and antiviral activities were evaluated. Their antiviral activity was assessed in model cell systems infected with human herpes simplex virus (HSV-1) and cytomegalovirus (CMV). Compounds DBA(1) and DBA(7) demonstrated in vitro inhibitory properties towards HSV-1, and DBA(7) completely blocked the viral infection. Compound DBA(11) displayed the in vitro therapeutic activity towards both HSV-1 and CMV. All of the DBA(n) could fluoresce, were well soluble in water, not cytotoxic to a concentration of 240⯵M, penetrated well into cell nuclei by binding to DNA and could inhibit topo-I at low micromolecular concentrations.
Assuntos
Antivirais/química , Benzimidazóis/química , DNA/química , Animais , Antivirais/síntese química , Antivirais/toxicidade , Benzimidazóis/síntese química , Benzimidazóis/toxicidade , Bovinos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citomegalovirus/efeitos dos fármacos , Fluorescência , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Ligantes , Solubilidade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/toxicidade , Células VeroRESUMO
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.
Assuntos
Bisbenzimidazol/análogos & derivados , DNA/química , DNA/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Bisbenzimidazol/síntese química , Bisbenzimidazol/farmacologia , Linhagem Celular , DNA/genética , DNA-Citosina Metilases/antagonistas & inibidores , Dimerização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Microscopia de Fluorescência , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologiaRESUMO
Dimeric bis-benzimidazoles (DBn) are the compounds specifically binding to A-T enriched sequences in the DNA minor groove. Due to this property they can inhibit DNA-dependent enzymes. We show that inhibition of the helicase activity of HCV NS3 protein by DBn was due to a novel mechanism, which involved direct binding of the ligands to the enzyme. The binding potency and inhibition efficacy depended on the length of the linker between the benzimidazole fragments. The most effective inhibitor DB11 partially prevented activation of NTPase activity of NS3 by poly(U) and increased affinity of the enzyme to the helicase substrate DNA.
Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Benzimidazóis/síntese química , Benzimidazóis/química , Dimerização , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development.
RESUMO
DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns+. The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5µM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters.
Assuntos
Aliivibrio fischeri/genética , Proteínas de Bactérias/genética , Bisbenzimidazol/farmacologia , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Percepção de Quorum/genética , Proteínas de Bactérias/antagonistas & inibidores , Composição de Bases/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Escherichia coli/genética , Medições Luminescentes , Photorhabdus/genéticaRESUMO
BACKGROUND: Hypermethylation is observed in the promoter regions of suppressor genes in the tumor cancer cells. Reactivation of these genes by demethylation of their promoters is a prospective strategy of the anticancer therapy. Previous experiments have shown that symmetric dimeric bisbenzimidazoles DBP(n) are able to block DNA methyltransferase activities. It was also found that DBP(n) produces a moderate effect on the activation of total gene expression in HeLa-TI population containing epigenetically repressed avian sarcoma genome. PRINCIPAL FINDINGS: It is shown that DBP(n) are able to penetrate the cellular membranes and accumulate in breast carcinoma cell MCF-7, mainly in the mitochondria and in the nucleus, excluding the nucleolus. The DBP(n) are non-toxic to the cells and have a weak overall demethylation effect on genomic DNA. DBP(n) demethylate the promoter regions of the tumor suppressor genes PTEN and RARB. DBP(n) promotes expression of the genes RARB, PTEN, CDKN2A, RUNX3, Apaf-1 and APC "silent" in the MCF-7 because of the hypermethylation of their promoter regions. Simultaneously with the demethylation of the DNA in the nucleus a significant increase in the methylation level of rRNA genes in the nucleolus was detected. Increased rDNA methylation correlated with a reduction of the rRNA amount in the cells by 20-30%. It is assumed that during DNA methyltransferase activity inhibition by the DBP(n) in the nucleus, the enzyme is sequestered in the nucleolus and provides additional methylation of the rDNA that are not shielded by DBP(n). CONCLUSIONS/SIGNIFICANCE: It is concluded that DBP (n) are able to accumulate in the nucleus (excluding the nucleolus area) and in the mitochondria of cancer cells, reducing mitochondrial potential. The DBP (n) induce the demethylation of a cancer cell's genome, including the demethylation of the promoters of tumor suppressor genes. DBP (n) significantly increase the methylation of ribosomal RNA genes in the nucleoli. Therefore the further study of these compounds is needed; it could lead to the creation of new anticancer agents.
Assuntos
Benzimidazóis/farmacologia , Metilação de DNA/efeitos dos fármacos , RNA Ribossômico/genética , Receptores do Ácido Retinoico/genética , Benzimidazóis/química , Dimerização , Células HeLa , Humanos , Células MCF-7 , PTEN Fosfo-Hidrolase , Espécies Reativas de Oxigênio/metabolismoRESUMO
In this study, a monomeric (MB) and a dimeric (DB) bisbenzimidazoles were identified as novel proteasome inhibitors of the trypsin-like activity located on ß2c sites of the constitutive 20S proteasome (IC50 values at 2-4 µM range). Remarkably, they were further shown to be 100- and 200-fold more potent inhibitors of the immunoproteasome trypsin-like activity (ß2i sites, IC50=24 nM) than of the homologous constitutive activity. Molecular models of inhibitor/enzyme complexes in the two types of trypsin-like sites and corresponding computed binding energy values corroborated kinetic data. Different binding modes were suggested for MB and DB to the ß2c and ß2i trypsic sites. Each pointed to better contacts of the ligand inside the ß2i active site than for ß2c site. MB and DB represent the first selective inhibitors of the immunoproteasome trypsin-like activity described to date and can be considered as prototypes for inhibiting this activity.