RESUMO
Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.
Assuntos
DNA/química , Conformação de Ácido Nucleico , RNA Polimerase II/metabolismo , Transcrição Gênica , Sequência de Bases , Imidazóis/química , Modelos Moleculares , Pirróis/química , Fatores de Elongação da Transcrição/metabolismoRESUMO
Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.
Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Microscopia Crioeletrônica , Reparo do DNA , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Transcrição Gênica , Adenosina Trifosfatases/química , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Domínios Proteicos , RNA Polimerase II/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The crucial role of androgen receptor (AR) in prostate cancer development is well documented, and its inhibition is a mainstay of prostate cancer treatment. Here, we analyze the perturbations to the AR cistrome caused by a minor groove binding molecule that is designed to target a sequence found in a subset of androgen response elements (ARE). We find treatment with this pyrrole-imidazole (Py-Im) polyamide exhibits sequence selectivity in its repression of AR binding in vivo. Differentially changed loci are enriched for sequences resembling ARE half-sites that match the Py-Im polyamide binding preferences determined in vitro. Comparatively, permutations of the ARE half-site bearing single or double mismatches to the Py-Im polyamide binding sequence are not enriched. This study confirms that the in vivo perturbation pattern caused by a sequence specific polyamide correlates with its in vitro binding preference genome-wide in an unbiased manner.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos/farmacologia , Imidazóis/farmacologia , Nylons/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Pirróis/farmacologia , Receptores Androgênicos/genética , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Expressão Gênica , Humanos , Imidazóis/química , Imidazóis/metabolismo , Masculino , Camundongos , Camundongos SCID , Nylons/química , Nylons/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Pirróis/química , Pirróis/metabolismo , Receptores Androgênicos/metabolismo , Elementos de Resposta , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The orphan nuclear receptors estrogen-related receptors (ERRs) bind to the estrogen-related receptor response element (ERRE) to regulate transcriptional programs in cellular metabolism and cancer cell growth. In this study, we evaluated the potential for a pyrrole-imidazole polyamide to block ERRα binding to ERREs to inhibit gene expression. We demonstrated that the ERRE-targeted polyamide 1 blocked the binding of ERRα to the consensus ERRE and reduced the transcriptional activity of ERRα in cell culture. We further showed that inhibiting ERRα transcriptional activity with polyamide 1 led to reduced mitochondrial oxygen consumption, a primary biological effect regulated by ERRα. Finally, our data demonstrated that polyamide 1 is an inhibitor for cancer cell growth.
RESUMO
RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a "minor groove sensor." Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy.
Assuntos
DNA/metabolismo , Nylons/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , DNA/química , DNA/genética , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico , Nylons/química , Nylons/farmacologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Pirróis/química , Pirróis/metabolismo , Pirróis/farmacologia , RNA Polimerase II/química , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos dos fármacosRESUMO
Means to cause an immunogenic cell death could lead to significant insight into how cancer escapes immune control. In this study, we screened a library of five pyrrole-imidazole polyamides coding for different DNA sequences in a model of B-cell lymphoma for the upregulation of surface calreticulin, a pro-phagocytosis signal implicated in immunogenic cell death. We found that hairpin polyamide 1 triggers the release of the damage-associated molecular patterns calreticulin, ATP and HMGB1 in a slow necrotic-type cell death. Consistent with this signaling, we observed an increase in the rate of phagocytosis by macrophages after the cancer cells were exposed to polyamide 1. The DNA sequence preference of polyamide 1 is 5'-WGGGTW-3' (where W = A/T), indicated by the pairing rules and confirmed by the Bind-n-Seq method. The close correspondence of this sequence with the telomere-repeat sequence suggests a potential mechanism of action through ligand binding at the telomere. This study reveals a chemical means to trigger an inflammatory necrotic cell death in cancer cells.
Assuntos
DNA/química , Linfoma de Células B/metabolismo , Fagocitose , Trifosfato de Adenosina/química , Animais , Calreticulina/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteína HMGB1/metabolismo , Humanos , Imidazóis/química , Immunoblotting , Inflamação , Células K562 , Luminescência , Macrófagos/citologia , Macrófagos/metabolismo , Necrose/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Nylons/química , Pirróis/química , Transdução de Sinais , Telômero/química , Regulação para CimaRESUMO
Pyrrole-imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.
Assuntos
Replicação do DNA/efeitos dos fármacos , Imidazóis/toxicidade , Nylons/toxicidade , Pirróis/toxicidade , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Estresse Fisiológico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Antígeno Nuclear de Célula em Proliferação/análise , Proteína de Replicação A/metabolismo , Estresse Fisiológico/genética , UbiquitinaçãoRESUMO
Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity.
Assuntos
Antineoplásicos/farmacologia , Nylons/farmacologia , Neoplasias da Próstata/prevenção & controle , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Imidazóis/química , Immunoblotting , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nylons/química , Nylons/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteassoma/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Pirróis/química , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Multidrug-resistant Staphylococcus aureus infections pose a significant threat to human health. Antibiotic resistance is most commonly propagated by conjugative plasmids like pLW1043, the first vancomycin-resistant S. aureus vector identified in humans. We present the molecular basis for resistance transmission by the nicking enzyme in S. aureus (NES), which is essential for conjugative transfer. NES initiates and terminates the transfer of plasmids that variously confer resistance to a range of drugs, including vancomycin, gentamicin, and mupirocin. The NES N-terminal relaxase-DNA complex crystal structure reveals unique protein-DNA contacts essential in vitro and for conjugation in S. aureus. Using this structural information, we designed a DNA minor groove-targeted polyamide that inhibits NES with low micromolar efficacy. The crystal structure of the 341-residue C-terminal region outlines a unique architecture; in vitro and cell-based studies further establish that it is essential for conjugation and regulates the activity of the N-terminal relaxase. This conclusion is supported by a small-angle X-ray scattering structure of a full-length, 665-residue NES-DNA complex. Together, these data reveal the structural basis for antibiotic multiresistance acquisition by S. aureus and suggest novel strategies for therapeutic intervention.
Assuntos
Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Biocatálise , DNA Bacteriano/genética , Modelos Moleculares , Nylons/farmacologia , Plasmídeos , Espalhamento a Baixo Ângulo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Difração de Raios XRESUMO
Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5'-WGGWWW-3' with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. An FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which was interrogated by quantitative reverse transcription-PCR and correlated with cell culture expression levels.
Assuntos
DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Imidazóis/farmacologia , Nylons/farmacologia , Pirróis/farmacologia , Transplante Heterólogo , Linhagem Celular Tumoral , Fluoresceína-5-Isotiocianato , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imidazóis/química , Imidazóis/metabolismo , Microscopia Confocal , Estrutura Molecular , Nylons/química , Nylons/metabolismo , Pirróis/química , Pirróis/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Técnicas de Síntese em Fase SólidaRESUMO
Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5'GGGWWW-3'. The compound is capable of binding to κB sites and reducing the expression of various NF-κB-driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α-inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists.
Assuntos
DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , NF-kappa B/metabolismo , Nylons/metabolismo , Transcrição Gênica/fisiologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Microscopia Confocal , NF-kappa B/antagonistas & inibidores , Desnaturação de Ácido Nucleico , Nylons/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Piridinas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5'-CGCG-3' and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImßIm-γ-PyImßIm (1), previously identified as a high affinity 5'-CGCG-3' binder, favors 5'-GCGC-3' in an unanticipated reverse binding orientation. Replacement of one ß alanine with Py to afford PyImPyIm-γ-PyImßIm (3) restores the preference for 5'-CGCG-3' binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA/genética , DNA/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/metabolismo , Imidazóis/farmacologia , Sequência de Bases , Ilhas de CpG , Metilação de DNA , Ensaios de Triagem em Larga Escala , Imidazóis/química , Especificidade por SubstratoRESUMO
In an effort to quantitate Py-Im polyamide concentrations in vivo, we synthesized the C-14 radioactively labeled compounds 1-3, and investigated their tumor localization in a subcutaneous xenograft model of prostate cancer (LNCaP). Tumor concentrations were compared with representative host tissues, and exhibited a certain degree of preferential localization to the xenograft. Compound accumulation upon repeated administration was measured. Py-Im polyamide 1 was found to accumulate in LNCaP tumors at concentrations similar to the IC50 value for this compound in cell culture experiments.
Assuntos
Imidazóis/farmacocinética , Nylons/farmacocinética , Neoplasias da Próstata/metabolismo , Pirróis/farmacocinética , Animais , Radioisótopos de Carbono/química , Xenoenxertos , Imidazóis/química , Imidazóis/metabolismo , Injeções Subcutâneas , Masculino , Camundongos , Estrutura Molecular , Transplante de Neoplasias , Nylons/química , Nylons/metabolismo , Neoplasias da Próstata/patologia , Pirróis/química , Pirróis/metabolismo , Distribuição TecidualRESUMO
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.
Assuntos
Imidazóis/química , Nylons/química , Pirróis/química , Transporte Biológico , Linhagem Celular , DNA/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Microscopia de Fluorescência , Nylons/síntese química , Nylons/metabolismo , Nylons/toxicidade , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA·TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA·TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA·TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA·TTC triplet repeats expanded at a rate of about two GAA·TTC triplet repeats/replication. However, GAA·TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA·TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA·TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA·TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system.
Assuntos
Reparo de Erro de Pareamento de DNA , Ataxia de Friedreich/metabolismo , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Modelos Biológicos , Proteína 2 Homóloga a MutS/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Diferenciação Celular/genética , Linhagem Celular , Fibroblastos/metabolismo , Fibroblastos/patologia , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Íntrons/genética , Proteínas de Ligação ao Ferro/genética , Fator 4 Semelhante a Kruppel , Camundongos , Proteína 2 Homóloga a MutS/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , FrataxinaRESUMO
Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC(50) values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.
Assuntos
Aminoácidos/química , Fluoresceína/química , Imidazóis/síntese química , Nylons/química , Nylons/síntese química , Pirróis/síntese química , Ácido gama-Aminobutírico/química , Linhagem Celular , Ciclização , Humanos , Imidazóis/química , Concentração Inibidora 50 , Micro-Ondas , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Pirróis/química , Técnicas de Síntese em Fase SólidaRESUMO
Groove specificity: pyrrole-imidazole polyamides are well-known for their specific interactions with the minor groove of DNA. However, polyamides do not show similar binding to duplex RNA, and a structural rationale for the molecular-level discrimination of nucleic acid duplexes by minor-groove-binding ligands is presented.
Assuntos
DNA/química , Imidazóis/química , Nylons/química , Pirróis/química , RNA/química , Sítios de Ligação , DNA/metabolismo , Imidazóis/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Nylons/metabolismo , Pirróis/metabolismo , RNA/metabolismoRESUMO
The pharmacokinetic properties of three pyrrole-imidazole (Py-Im) polyamides of similar size and Py-Im content but different shape were studied in the mouse. Remarkably, hairpin and cyclic oligomers programmed for the same DNA sequence 5'-WGGWWW-3' displayed distinct pharmacokinetic properties. Furthermore, the hairpin 1 and cycle 2 exhibited vastly different animal toxicities. These data provide a foundation for design of DNA binding Py-Im polyamides to be tested in vivo.
Assuntos
Imidazóis/química , Imidazóis/farmacocinética , Nylons/química , Nylons/farmacocinética , Pirróis/química , Pirróis/farmacocinética , Animais , Peso Corporal/efeitos dos fármacos , Imidazóis/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Nylons/efeitos adversos , Pirróis/efeitos adversosRESUMO
Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing ß/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.
Assuntos
DNA/química , Imidazóis/química , Nylons/química , Pirróis/química , DNA/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
We have shown that a specific pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugate, 1R-Chl, alters the growth characteristics of various cancer cell lines in culture, and causes these cells to arrest in the G2/M stage of the cell cycle, without apparent cytotoxicity. This molecule has also shown efficacy in several mouse xenograft models, preventing tumor growth. Previous microarray studies have suggested that members of the histone H4 gene family, H4c and H4j/k, are the primary targets of this molecule, leading to reduced histone mRNA synthesis and growth arrest in cancer cells. In the present study, we examine the effects of 1R-Chl on transcription of other members of the H4 gene family, with the result that mRNA transcription of most genomic copies of H4 are down-regulated by 1R-Chl in a human pancreatic cancer cell line (MIA PaCa-2), but not in a cell line of non-cancerous origin (HEK293 cells). The basis for this differential effect is likely an open chromatin conformation within the H4 genes in cancer cells. Chromatin immunoprecipitation experiments show increased histone acetylation on the histone H4 genes in cancer cells, compared to HEK293 cells, explaining the differential activity of this molecule in cancer versus non-cancer cells.