RESUMO
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 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
Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl-aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/química , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/química , Triazóis/síntese química , Triazóis/farmacologia , Técnicas de Química Sintética , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Solubilidade , Relação Estrutura-Atividade , Triazóis/química , Água/químicaRESUMO
BACKGROUND: Macrophage migration inhibitory factor (MIF) exerts a protective effect on ischemic myocardium by activating AMP-activated protein kinase (AMPK). Small molecules that increase the affinity of MIF for its receptor have been recently designed, and we hypothesized that such agonists may enhance AMPK activation and limit ischemic tissue injury. METHODS AND RESULTS: Treatment of cardiomyocytes with the candidate MIF agonist, MIF20, augmented AMPK phosphorylation, increased by 50% the surface localization of glucose transporter, and enhanced by 25% cellular glucose uptake in comparison with MIF alone. In mouse hearts perfused with MIF20 before no-flow ischemia and reperfusion, postischemic left ventricular function improved commensurately with an increase in cardiac MIF-AMPK activation and an augmentation in myocardial glucose uptake. By contrast, small-molecule MIF agonism was not effective in cells or tissues genetically deficient in MIF or the MIF receptor, verifying the specificity of MIF20 for MIF-dependent AMPK signaling. The protective effect of MIF20 also was evident in an in vivo regional ischemia model. Mice treated with MIF20 followed by left coronary artery occlusion and reperfusion showed a significant reduction in infarcted myocardium. CONCLUSIONS: These data support the pharmacological utility of small-molecule MIF agonists in enhancing AMPK activation and reducing cardiac ischemic injury.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Histocompatibilidade Classe II/genética , Oxirredutases Intramoleculares/farmacologia , Fatores Inibidores da Migração de Macrófagos/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Cardiotônicos/farmacologia , Células Cultivadas , Glucose/farmacocinética , Antígenos de Histocompatibilidade Classe II/metabolismo , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/genética , Isoxazóis/farmacologia , Fatores Inibidores da Migração de Macrófagos/agonistas , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: The role and mechanism of action of MIF in bronchopulmonary dysplasia (BPD) are not known. We hypothesized that increased MIF signaling would ameliorate the pulmonary phenotype of BPD in the mouse lung. METHODS: We studied newborn wild type (WT), MIF knockout (MIFKO), and lung MIF transgenic (MIFTG) mice in room air and a BPD model, and examined the effects of administering a small molecule MIF agonist and antagonist. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed. RESULTS: The pulmonary phenotype of MIFKO and MIFTG mice lungs in room air (RA) and BPD model were comparable to the WT-BPD mice at postnatal (PN) day 14. Vascular endothelial growth factor (VEGF)-A, -R1 and Angiopoietin (Ang)1 mRNA were decreased, and Ang2 increased in the WT-BPD, MIFKO-RA, MIFKO-BPD, MIFTG-RA and MIFTG-BPD mice lungs, compared to appropriate controls. The protein expression of Ang1 in the MIFKO-RA was similar to WT-RA, but decreased in MIFTG-RA, and decreased in all the BPD groups. Ang2 was increased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. Tie2 was increased in WT-BPD compared to WT-RA, but decreased in MIFKO- and MIFTG- RA and BPD groups. VEGFR1 was uniformly decreased in MIFKO-RA, MIFTG-RA and in all 3 BPD groups. VEGF-A had a similar expression across all RA and BPD groups. There was partial recovery of the pulmonary phenotype in the WT-BPD model treated with the MIF agonist, and in the MIFTG mice treated with the MIF antagonist. CONCLUSIONS: These data point to the careful regulatory balance exerted by MIF in the developing lung and response to hyperoxia and support the potential therapeutic value of small molecule MIF modulation in BPD.
Assuntos
Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/metabolismo , Fatores Quimiotáticos/metabolismo , Hiperóxia/complicações , Hiperóxia/metabolismo , Pulmão/metabolismo , Macrófagos/imunologia , Animais , Animais Recém-Nascidos , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Regulação para CimaRESUMO
Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds were found to possess antagonist activity in the low micromolar range with the most potent being the 6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.
Assuntos
Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Tiazóis/química , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Simulação por Computador , Desenho de Fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Fatores Inibidores da Migração de Macrófagos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Tiazóis/síntese química , Tiazóis/farmacologiaRESUMO
The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto-enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1 µM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC(50) values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF-CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts.
Assuntos
Benzoxazóis/química , Receptores Imunológicos/antagonistas & inibidores , Antígenos de Diferenciação de Linfócitos B/química , Benzoxazóis/síntese química , Benzoxazóis/farmacologia , Sítios de Ligação , Simulação por Computador , Fibroblastos/enzimologia , Antígenos de Histocompatibilidade Classe II/química , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Receptores Imunológicos/metabolismoRESUMO
The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also promotes AMPK activation with potential benefits for response to myocardial infarction and ischemia-reperfusion. Structure-based molecular design has led to the discovery of not only antagonists, but also the first agonists of MIF-CD74 binding. The compounds contain a triazole core that is readily assembled via Cu-catalyzed click chemistry. The agonist and antagonist behaviors were confirmed via study of MIF-dependent ERK1/2 phosphorylation in human fibroblasts.
Assuntos
Desenho de Fármacos , Receptores Imunológicos/agonistas , Triazóis/farmacologia , Células Cultivadas , Química Click , Cobre , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress.
Assuntos
Nylons/toxicidade , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/química , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases/química , DNA Topoisomerases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/química , Masculino , Camundongos , Nylons/síntese química , Nylons/química , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Pirróis/química , Receptores Androgênicos/metabolismo , Análise de Sequência de RNA , Transplante HeterólogoRESUMO
The Plasmodium falciparum orthologue of the human cytokine, macrophage migratory inhibitory factor (PfMIF), is produced by the parasite during malaria infection and modulates the host's immune response. As for other MIF orthologues, PfMIF has tautomerase activity, whose inhibition may influence the cytokine activity. To identify small-molecule inhibitors of the tautomerase activity of PfMIF, virtual screening has been performed by docking 2.1 million compounds into the enzymatic site. Assaying of 17 compounds identified four as active. Substructure search for the most potent of these compounds, a 4-phenoxypyridine analogue, identified four additional compounds that were purchased and also shown to be active. Thirty-one additional analogues were then designed, synthesized, and assayed. Three were found to be potent PfMIF tautomerase inhibitors with K(i) of â¼40 nM; they are also highly selective with K(i) > 100 µM for human MIF.
Assuntos
Antimaláricos/farmacologia , Ensaios de Triagem em Larga Escala , Isomerases/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Antimaláricos/química , Humanos , Ligação de Hidrogênio , Malária/metabolismo , Malária/parasitologia , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Resonant two-photon ionization (R2PI), resonant ion-dip infrared (RIDIR), and UV-UV hole-burning spectroscopies have been employed to obtain conformation-specific infrared and ultraviolet spectra under supersonic expansion conditions for O-(2-acetamidoethyl)-N-acetyltyramine (OANAT), a doubly substituted aromatic in which amide-containing alkyl and alkoxy side chains are located in para positions on a phenyl ring. For comparison, three single-chain analogs were also studied: (i) N-phenethyl-acetamide (NPEA), (ii) N-(p-methoxyphenethyl-acetamide) (NMPEA), and (iii) N-(2-phenoxyethyl)-acetamide (NPOEA). Six conformations of OANAT have been resolved, with S(0)-S(1) origins ranging from 34,536 to 35,711 cm(-1), denoted A-F, respectively. RIDIR spectra show that conformers A-C each possess an intense, broadened amide NH stretch fundamental shifted below 3400 cm(-1), indicative of the presence of an interchain H bond, while conformers D-F have both amide NH stretch fundamentals in the 3480-3495 cm(-1) region, consistent with independent-chain structures with two free NH groups. NPEA has a single conformer with S(0)-S(1) origin at 37,618 cm(-1). NMPEA has three conformers, two that dominate the R2P1 spectrum, with origin transitions between 35,580 and 35,632 cm(-1). Four conformations, one dominate and three minor, of NPOEA have been resolved with origins between 35,654 and 36,423 cm(-1). To aid the making of conformational assignments, the geometries of low-lying structures of all four molecules have been optimized and the associated harmonic vibrational frequencies calculated using density functional theory (DFT) and RIMP2 methods. The S(0)-S(1) adiabatic excitation energies have been calculated using the RICC2 method and vertical excitation energies using single-point time-dependent DFT. The sensitivity of the S(0)-S(1) energy separation in OANAT and NPOEA primarily arises from different orientations of the chain attached to the phenoxy group. Using the results of the single-chain analogs, tentative assignments have been made for the observed conformers of OANAT. The RIMP2 calculations predict that interchain H-bonded conformers of OANAT are 25-30 kJ/mol more stable than the extended-chain structures. However, the free energies of the interchain H-bonded and extended structures calculated at the preexpansion temperature (450 K) differ by less than 10 kJ/mol, and the number of extended structures far outweighs the number of H-bonded conformers. This entropy-driven effect explains the presence of the independent-chain conformers in the expansion, and cautions future studies that rely solely on relative energies of conformers in considering possible assignments.