RESUMO
BACKGROUND AND PURPOSE: Non-alcoholic hepatic fatty liver disease (NAFLD) is a manifestation of the metabolic syndrome in the liver and non-alcoholic steatohepatitis (NASH) represents its advanced stage. R17 derived from bouchardatine, shows benefits in the metabolic syndrome, but has not been tested in the liver. The present study examined the pharmacological effects of R17 in a model of NAFLD/NASH and its mode of action. EXPERIMENTAL APPROACH: The effects of R17 were examined in mice fed a high-fat (HF) diet to induce the pathological characteristics of NAFLD/NASH and in cultures of HuH7 cells. We used histological and immunohistochemical techniques along with western blotting and siRNA. Generation of ROS and apoptosis were measured. KEY RESULTS: Administration of R17 (20 mg·kg-1 , i.p. every other day) for 5 weeks reversed HF-induced hepatic triglyceride content, inflammation (inflammatory cytokines and macrophage numbers), injury (hepatocyte ballooning and apoptosis, plasma levels of alanine aminotransferase and aspartate aminotransferase), and fibrogenesis (collagen deposition and mRNA expression of fibrosis markers). In cultured cells, R17 reduced cell steatosis from both lipogenesis and fatty acid influx. The attenuated inflammation and cell injury were associated with inhibition of both endoplasmic reticulum (ER) stress and oxidative stress. Notably, R17 activated the liver kinase B1-AMP-activated protein kinase (AMPK) pathway by inhibiting activity of ATP synthase, rather than direct stimulation of AMPK. CONCLUSION AND IMPLICATIONS: R17 has therapeutic potential for NAFLD/NASH. Its mode of action involves the elimination of ER and oxidative stresses, possibly via activating the LKB1-AMPK axis by inhibiting the activity of ATP synthase.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Alcaloides Indólicos/farmacologia , Alcaloides Indólicos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo/efeitos dos fármacos , Triglicerídeos/metabolismoRESUMO
Our previous studies reveal that indolizinoquinolinedione scaffold is a base to develop novel DNA topoisomerase IB (TOP1) catalytic inhibitors. In this work, twenty-three novel indolizinoquinolinedione derivatives were synthesized. TOP1-mediated relaxation, nicking and unwinding assays revealed that three fluorinated derivatives 26, 28 and 29, and one N,N-trans derivative 46 act as TOP1 catalytic inhibitors with higher TOP1 inhibition (++++) than camptothecin (+++) and without TOP1-mediated unwinding effect. MTT assay against five human cancer cell lines indicated that the highest cytotoxicity is 20 for CCRF-CEM cells, 25 for A549 and DU-145â¯cells, 26 for HCT116â¯cells, and 33 for Huh7 cells with GI50 values at nanomolar range. The drug-resistant cell assay indicated that compound 26 may mainly act to TOP1 in cells and are less of Pgp substrates. Flow cytometric analysis showed that compounds 26, 28 and 29 can obviously induce apoptosis of HCT116â¯cells. Moreover, the structure-activity relationship (SAR) of indolizinoquinolinedione derivatives was analyzed.
Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Indolizinas/farmacologia , Quinolonas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Biocatálise , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indolizinas/síntese química , Indolizinas/química , Estrutura Molecular , Quinolonas/síntese química , Quinolonas/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Células Tumorais CultivadasRESUMO
BACKGROUND: Telomeric repeat-containing RNA (TERRA) is a large non-coding RNA in mammalian cells, which forms an integral component of telomeric heterochromatin. TERRA can bind to an allosteric site of telomeric repeat factor 2 (TRF2), a key component of Shelterin that protect chromosome termini. Both TERRA and TRF2 have been recognized as promising new therapeutic targets for cancer treatment. METHODS: Our methods include FRET assay, SPR, CD, microscale thermophoresis (MST), enzyme-linked immunosorbent assay (ELISA), chromatin immunoprecipitation (ChIP), colony formation assays, Western blot, immunofluorescence, cell cycle arrest and apoptosis detection, and xCELLigence real-time cell analysis (RTCA). RESULTS: In our routine screening of small molecule libraries, we found that a Quindoline derivative, CK1-14 could bind to and stabilize TERRA G-quadruplex structure, which could bind more tightly with an allosteric site of a telomeric binding protein TRF2, resulting in dissociation of TRF2 from telomeric DNA. Further in cellular studies indicated that the above effect of CK1-14 on TERRA G-quadruplex could activate DNA-damage response and cause cell cycle arrest, resulting in inhibition of U2OS cell proliferation and causing cell apoptosis. CONCLUSIONS: Our mechanistic studies indicated that interaction of CK1-14 with TERRA induces telomeric DNA-damage response in U2OS cancer cells through inhibition of TRF2. CK1-14 could be further developed as a promising lead compound targeting telomere for cancer treatment. GENERAL SIGNIFICANCE: Our present study provides the first evidence that allosteric modulation of TRF2 by TERRA G-quadruplex with a binding ligand could become a promising new strategy for cancer treatment especially for ALT tumor cells.
Assuntos
Alcaloides/farmacologia , Dano ao DNA , Indóis/farmacologia , Neoplasias/tratamento farmacológico , Quinolinas/farmacologia , RNA Longo não Codificante/metabolismo , Telômero , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Alcaloides/metabolismo , Regulação Alostérica , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quadruplex G , Humanos , Indóis/metabolismo , Neoplasias/genética , Neoplasias/patologia , Quinolinas/metabolismoRESUMO
Telomeric repeat factor 2 (known as TRF2 or TERF2) is a key component of telomere protection protein complex named as Shelterin. TRF2 helps the folding of telomere to form T-loop structure and the suppression of ATM-dependent DNA damage response activation. TRF2 has been recognized as a potentially new therapeutic target for cancer treatment. In our routine screening of small molecule libraries, we found that Curcusone C had significant effect in disrupting the binding between TRF2 and telomeric DNA, with potent antitumor activity against cancer cells. Our result showed that Curcusone C could bind with TRF2 without binding interaction with TRF1 (telomeric repeat factor 1) although these two proteins share high sequence homology, indicating that their binding conformations and biological functions in telomere could be different. Our mechanistic studies showed that Curcusone C bound with TRF2 possibly through its DNA binding site causing blockage of its interaction with telomeric DNA. Further in cellular studies indicated that the interaction of TRF2 with Curcusone C could activate DNA-damage response, inhibit tumor cell proliferation, and cause cell cycle arrest, resulting in tumor cell apoptosis. Our studies showed that Curcusone C could become a promising lead compound for further development for cancer treatment. Here, TRF2 was firstly identified as a target of Curcusone C. It is likely that the anti-cancer activity of some other terpenes and terpenoids are related with their possible effect for telomere protection proteins.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , DNA de Neoplasias/genética , Diterpenos/farmacologia , Regulação Neoplásica da Expressão Gênica , Telômero/efeitos dos fármacos , Proteína 2 de Ligação a Repetições Teloméricas/genética , Antineoplásicos Fitogênicos/isolamento & purificação , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Diterpenos/isolamento & purificação , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células HeLa , Humanos , Especificidade de Órgãos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Telômero/química , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
c-MYC is one of the important human proto-oncogenes, and transcriptional factor NM23-H2 can activate c-MYC transcription by recognizing the G-quadruplex in the promoter of the gene. Small molecules that inhibit c-MYC transcription by disrupting the NM23-H2/G-quadruplex interaction might be a promising strategy for developing selective anticancer agents. In recent studies, we developed a series of isaindigotone derivatives, which can bind to G-quadruplex and NM23-H2, thus down-regulating c-MYC ( J. Med. Chem. 2017 , 60 , 1292 - 1308 ). Herein, a series of novel isaindigotone derivatives were designed, synthesized, and screened for NM23-H2 selective binding ligands. Among them, compound 37 showed a high specific binding affinity to NM23-H2, effectively disrupting the interaction of NM23-H2 with G-quadruplex, and it strongly down-regulated c-MYC transcription. Furthermore, 37 induced cell cycle arrest and apoptosis, and it exhibited good tumor growth inhibition in a mouse xenograft model. This work provides a new strategy to modulate c-MYC transcription for the development of selective anticancer drugs.
Assuntos
Antineoplásicos/farmacologia , Quadruplex G , Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Pirróis/farmacologia , Quinazolinas/farmacologia , Quinazolinonas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Doxorrubicina/farmacologia , Desenho de Fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Ligantes , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Nucleosídeo NM23 Difosfato Quinases/química , Nucleosídeo NM23 Difosfato Quinases/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Pirróis/administração & dosagem , Pirróis/síntese química , Pirróis/química , Quinazolinas/administração & dosagem , Quinazolinas/síntese química , Quinazolinas/química , Quinazolinonas/administração & dosagem , Quinazolinonas/síntese química , Quinazolinonas/química , Relação Estrutura-Atividade , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The c-MYC oncogene is overactivated during Burkitt's lymphoma pathogenesis. Targeting c-MYC to inhibit its transcriptional activity has emerged as an effective anticancer strategy. We synthesized four series of disubstituted quindoline derivatives by introducing the second cationic amino side chain and 5-N-methyl group based on a previous study of SYUIQ-5 (1) as c-MYC promoter G-quadruplex ligands. The in vitro evaluations showed that all new compounds exhibited higher stabilities and binding affinities, and most of them had better selectivity (over duplex DNA) for the c-MYC G-quadruplex compared to 1. Moreover, the new ligands prevented NM23-H2, a transcription factor, from effectively binding to the c-MYC G-quadruplex. Further studies showed that the selected ligand, 7a4, down-regulated c-MYC transcription by targeting promoter G-quadruplex and disrupting the NM23-H2/c-MYC interaction in RAJI cells. 7a4 could inhibit Burkitt's lymphoma cell proliferation through cell cycle arrest and apoptosis and suppress tumor growth in a human Burkitt's lymphoma xenograft.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Indóis/farmacologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Quinolinas/farmacologia , Alcaloides/síntese química , Alcaloides/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G/efeitos dos fármacos , Humanos , Indóis/síntese química , Indóis/química , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Promoting energy metabolism is known to provide therapeutic effects for obesity and associated metabolic disorders. The present study evaluated the therapeutic effects of the newly identified bouchardatine (Bou) on obesity-associated metabolic disorders and the molecular mechanisms of these effects. EXPERIMENTAL APPROACH: The molecular mode of action of Bou for its effects on lipid metabolism was first examined in 3T3-L1 adipocytes and HepG2 cells. This was followed by an evaluation of its metabolic effects in mice fed a high-fat diet for 16 weeks with Bou being administered in the last 5 weeks. Further mechanistic investigations were conducted in pertinent organs of the mice and relevant cell models. KEY RESULTS: In 3T3-L1 adipocytes, Bou reduced lipid content and increased sirtuin 1 (SIRT1) activity to facilitate liver kinase B1 (LKB1) activation of AMPK. Chronic administration of Bou (50 mgâkg-1 every other day) in mice significantly attenuated high-fat diet-induced increases in body weight gain, dyslipidaemia and fatty liver without affecting food intake and no adverse effects were detected. These metabolic effects were associated with activation of the SIRT1-LKB1-AMPK signalling pathway in adipose tissue and liver. Of particular note, UCP1 expression and mitochondrial biogenesis were increased in both white and brown adipose tissues of Bou-treated mice. Incubation with Bou induced similar changes in primary brown adipocytes isolated from mice. CONCLUSIONS AND IMPLICATIONS: Bou may have therapeutic potential for obesity-related metabolic diseases by increasing the capacity of energy expenditure in adipose tissues and liver through a mechanism involving the SIRT1-LKB1-AMPK axis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Produtos Biológicos/farmacologia , Alcaloides Indólicos/farmacologia , Doenças Metabólicas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Sirtuína 1/metabolismo , Células 3T3-L1 , Animais , Produtos Biológicos/administração & dosagem , Dieta Hiperlipídica , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Alcaloides Indólicos/administração & dosagem , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The specificity of nucleic acids' binders is crucial for developing this kind of drug, especially for novel G-quadruplexes' binders. Quindoline derivatives have been developed as G-quadruplex stabilizers with good interactive activities. In order to improve the selectivity and binding affinity of quindoline derivatives as c-myc G-quadruplex binding ligands, novel triazole containing benzofuroquinoline derivatives (T-BFQs) were designed and synthesized by using the 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks. The selectivity toward c-myc G-quadruplex DNA of these novel T-BFQs was significantly improved, together with an obvious increase on binding affinity. Further cellular and in vivo experiments indicated that the T-BFQs showed inhibitory activity on tumor cells' proliferation, presumably through the down-regulation of transcription of c-myc gene. Our findings broadened the modification strategies of specific G-quadruplex stabilizers.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Quadruplex G/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Quinolonas/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Química Click , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinolonas/síntese química , Quinolonas/química , Relação Estrutura-Atividade , Termodinâmica , Triazóis/químicaRESUMO
In our previous research, 9-bromo indolizinoquinoline-5,12-dione 1 has been found to be a good anti-MRSA agent. However, it had very low bioavailability in vivo possibly due to its low solubility in water. In order to obtain the derivatives with higher anti-MRSA activity and good water solubility, twenty eight bromo-substituted indolizinoquinoline-5,12-dione derivatives were synthesized in the present study. The antibacterial activity of the synthesized compounds was evaluated against one gram-negative and some gram-positive bacterial strains including 100 clinical MRSA strains. The UV assays were carried out to determine the solubility of six active compounds 16, 21, 23 and 27-29. The most potent compound 28 exhibited strong activity against clinical MRSA strains with both MIC50 and MIC90 values lower than 7.8 ng/mL. Compound 27 had good water solubility of 1.98 mg/mL and strong activity against clinical MRSA strains with MIC50 value of 63 ng/mL and MIC90 value of 125 ng/mL, 16-fold higher than that of Vancomycin.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Quinolinas/síntese química , Quinolinas/farmacologia , Antibacterianos/química , Bactérias/efeitos dos fármacos , Técnicas de Química Sintética , Humanos , Testes de Sensibilidade Microbiana , Quinolinas/química , Solubilidade , Água/químicaRESUMO
Transcriptional control of c-myc oncogene is an important strategy for antitumor drug design. G-quadruplexes in the promoter region have been proven to be the transcriptional down-regulator of this gene. The transcriptional factor NM23-H2 can reactivate c-myc transcription by unwinding the G-quadruplex structure. Thus, down-regulation of c-myc transcription via disrupting G-quadruplex-NM23-H2 interaction might be a potential approach for cancer therapy. Here, a series of new isaindigotone derivatives were designed and synthesized based on our previous study. The abilities of these derivatives on interacting with G-quadruplexes or NM23-H2, and disrupting G-quadruplex-NM23-H2 interaction were evaluated. Among these derivatives, 19d and 22d showed remarkable abilities on disrupting G-quadruplex-NM23-H2 interaction. They exhibited significant effects on c-myc-relating processes in SiHa cells, including inhibiting the transcription and translation, inhibiting cellular proliferation, inducing apoptosis, and regulating cell cycle. Our findings provided the basis for the anticancer strategy based on c-myc transcriptional regulation via small molecules disrupting G-quadruplex-protein interaction.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Quinazolinas/farmacologia , Alcaloides/síntese química , Alcaloides/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Quinazolinas/síntese química , Quinazolinas/química , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
IZNP-1: Multiple G-quadruplex units in the 3Î-terminal overhang of human telomeric DNA can associate and form multimeric structures. The specific targeting of such distinctive higher-order G-quadruplexes might be a promising strategy for developing selective anticancer agents with fewer side effects. However, thus far, only a few molecules were found to selectively bind to telomeric multimeric G-quadruplexes, and their effects on cancer cells were unknown. In this study, a new triaryl-substituted imidazole derivative called was synthesized and found to specifically bind to and strongly stabilize telomeric multimeric G-quadruplexes through intercalating into the pocket between the two quadruplex units. The pocket size might affect the binding behavior of . Further cellular studies indicated that could provoke cell cycle arrest, apoptosis and senescence in Siha cancer cells, mainly because of telomeric DNA damage and telomere dysfunction induced by the interactions of with telomeric G-quadruplexes. Notably, had no effect on the transcriptional levels of several common oncogenes that have the potential to form monomeric G-quadruplex structures in their promoter regions. Such behavior differed from that of traditional telomeric G-quadruplex ligands. Accordingly, this work provides new insights for the development of selective anticancer drugs targeting telomeric multimeric G-quadruplexes.
Assuntos
Quadruplex G , Imidazóis/química , Telômero/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/química , Dano ao DNA/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Oncogenes/genética , Análise Espectral , Telômero/genética , Transcrição GênicaRESUMO
G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (<3 nt). By using D1, telomeric parallel G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo.
Assuntos
Quadruplex G , Anticorpos de Cadeia Única/química , Telômero/química , Sequência de Bases , Linhagem Celular , Sequência Consenso , Genoma Humano , Células HeLa , Humanos , Ligantes , Modelos Moleculares , Anticorpos de Cadeia Única/imunologia , Telômero/genética , Telômero/imunologiaRESUMO
The RNA G-quadruplex is an important secondary structure formed by guanine-rich RNA sequences. However, its folding studies have mainly been studied in vitro. Accurate identification of RNA G-quadruplex formation within a sequence of interest remains difficult in cells. Herein, and based on the guanine-rich sequence in the 5'-UTR of NRAS mRNA, we designed and synthesized the first G-quadruplex-triggered fluorogenic hybridization (GTFH) probe, ISCH-nras1, for the unique visualization of the G-quadruplexes that form in this region. ISCH-nras1 is made up of two parts: The first is a fluorescent light-up moiety specific to G-quadruplex structures, and the second is a DNA molecule that can hybridize with a sequence that is adjacent to the guanine-rich sequence in the NRAS mRNA 5'-UTR. Further evaluation studies indicated that ISCH-nras1 could directly and precisely detect the targeted NRAS RNA G-quadruplex structures, both in vitro and in cells. Thus, this GTFH probe was a useful tool for directly investigating the folding of G-quadruplex structures within an RNA of interest and represents a new direction for the design of smart RNA G-quadruplex probes.
Assuntos
Engenharia , Corantes Fluorescentes/metabolismo , Quadruplex G , GTP Fosfo-Hidrolases/genética , RNA/química , RNA/metabolismo , Sequência de Bases , Hibridização de Ácido NucleicoRESUMO
G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G-quadruplex-forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Quadruplex G , Imidazóis/química , Colorimetria/métodos , DNA/química , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Fluorescência/métodosRESUMO
Novel topoisomerase II (Topo II) inhibitors have gained considerable interest for the development of anticancer agents. In this study, a series of 1,3-benzoazolyl-substituted pyrrolo[2,3-b]pyrazine derivatives were designed, synthesized, and evaluated as potential Topo II catalytic inhibitors. It was found that some of derivatives had good antiproliferative activity on seven cancer cell lines, especially on HL-60/MX2, a cancer cell line derivative from HL-60 that is resistant to Topo II poison. Topo II mediated DNA relaxation assay results showed that derivatives could significantly inhibit the activity of Topo II, and the structure-activity relationship studies indicated the importance of the alkylamino side chain and the benzoazolyl group. Further mechanism studies revealed that derivatives function as Topo II nonintercalative catalytic inhibitors and may block the ATP binding site of Topo II. Moreover, flow cytometric analysis showed that this class of compounds could induce apoptosis of HL-60 cells.
Assuntos
Pirazinas/síntese química , Pirazinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Catálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , DNA Helicases/efeitos dos fármacos , DNA Topoisomerases Tipo II/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Relação Estrutura-AtividadeRESUMO
Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology.
Assuntos
Corantes Fluorescentes/química , Quadruplex G , Aspartato-tRNA Ligase/química , Aspartato-tRNA Ligase/metabolismo , Sítios de Ligação , Química Click , Simulação de Acoplamento Molecular , Ácidos Nucleicos/química , Estrutura Terciária de Proteína , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Espectrometria de FluorescênciaRESUMO
Our recent study has shown that the natural product bouchardatine (1) can reduce the triglyceride (TG) content in 3T3-L1 adipocytes (EC50 ≈ 25 µM). Here, we synthesized two series of compounds by introducing amine side chains at the 5 or 8 position of 1 and evaluated the lipid-lowering activity of derivatives. It was found that some of the compounds had significant lipid-lowering effects, and the most active compound 3d showed better activity (EC50 = 0.017 µM) than 2 (EC50 = 0.086 µM), a compound reported by us. Further, the mechanism studies revealed that 3d blocked TG accumulation via activation of the LKB1-AMPK signaling pathway, efficiently down-regulating the expression of key regulators of adipogenesis/lipogenesis. Cell uptake assay and confocal imaging of 3d in cells indicated that compound 3d had favorable cell permeability. Our results suggest that 3d may be a promising agent for the treatment of obesity and related metabolic disorders.
Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Alcaloides Indólicos/química , Alcaloides Indólicos/farmacologia , Lipogênese/efeitos dos fármacos , Quinazolinas/química , Quinazolinas/farmacologia , Triglicerídeos/metabolismo , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacocinética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Colesterol/metabolismo , Células Hep G2 , Humanos , Alcaloides Indólicos/síntese química , Alcaloides Indólicos/farmacocinética , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Transdução de Sinais/efeitos dos fármacosRESUMO
The guanine-rich sequences are able to fold into G-quadruplexes in living cells, making these structures promising anti-cancer drug targets. In the current study, we identified a small molecule, Ber8, from a series of 9-substituted berberine derivatives and found that it could induce acute cell growth arrest and senescence in cancer cells, but not in normal fibroblasts. Further analysis revealed that the cell growth arrest was directly associated with apparent cell cycle arrest, cell senescence, and profound DNA damage at the telomere region. Significantly, our studies also provided evidence that Ber8 could stabilize endogenous telomeric G-quadruplexes structures in cells. Ber8 could then induce the delocalization of TRF1 and POT1 from the telomere accompanied by a rapid telomere uncapping. These results provide compelling insights into direct binding of telomeric G-quadruplexes and might contribute to the development of more selective, effective anticancer drugs.
Assuntos
Anticarcinógenos/farmacologia , Berberina/farmacologia , Carcinoma/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Anticarcinógenos/síntese química , Berberina/análogos & derivados , Berberina/síntese química , Carcinoma/patologia , Ciclo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Fibroblastos/fisiologia , Células HL-60 , Humanos , Complexo Shelterina , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
The promoter of hnRNP K oncogene was found to contain a G/C-rich sequence on the same DNA strand, which can form interconvertible G-quadruplex, i-motif, and hairpin structures. Protein CNBP could bind and stabilize the G-quadruplex, inducing transformation of the hairpin into the G-quadruplex, resulting in down-regulation of hnRNP K transcription. In contrast, Corticosterone could bind and stabilize the hairpin, inducing transformation of the G-quadruplex into the hairpin, resulting in up-regulation of hnRNP K gene transcription.
Assuntos
DNA/química , Quadruplex G , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Sequência de Bases , Citosina/química , Guanina/química , Estrutura Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Elementos Reguladores de TranscriçãoRESUMO
In our previous work, indolizinoquinolinedione derivative 1 was identified as a Top1 catalytic inhibitor. Herein, a series of 6-substituted indolizinoquinolinedione derivatives were synthesized through modification of the parent compound 1. Top1 cleavage and relaxation assays indicate that none of these novel compounds act as classical Top1 poison, and that the compounds with alkylamino terminus at C-6 side chain, including 8, 11-16, 18-21, 25, 26 and 28-30, are the most potent Top1 catalytic inhibitors. Top1-mediated unwinding assay demonstrated that 14, 22 and 26 were Top1 catalytic inhibitors without Top1-mediated unwinding effect. Moreover, MTT results showed that compounds 26, 28-30 exhibit significant cytotoxicity against human leukemia HL-60 cells, and that compound 26 exerts potent cytotoxicity against A549 lung cancer cells at nanomolar range.