RESUMO
DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging. We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity. The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures. The ligand-G4 interaction studied with 1H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures. Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.
Assuntos
Antineoplásicos/química , Neoplasias da Mama , Quadruplex G , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Desenho de Fármacos , Feminino , Genes myc , Humanos , Ligantes , Células MCF-7 , Camundongos , Regiões Promotoras Genéticas , TelômeroRESUMO
The study on the mechanism and kinetics of mRNA degradation provides a new vision for chemical intervention on protein expression. The AU enrichment element (ARE) in mRNA 3'-UTR can be recognized and bound by the ARE binding protein (AU-rich Element factor (AUF1) to recruit RNase for degradation. In the present study, we proposed a novel strategy for expression regulation that interferes with the AUF1-RNA binding. A small-molecule compound, JNJ-7706621, was found to bind AUF1 protein and inhibit mRNA degradation by screening the commercial compound library. We discovered that JNJ-7706621 could inhibit the expression of AUF1 targeted gene IL8, an essential pro-inflammatory factor, by interfering with the mRNA homeostatic state. These studies provide innovative drug design strategies to regulate mRNA homeostasis.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Regiões 3' não Traduzidas , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Protein synthesis is critical to cell survival and translation regulation is essential to post-transcriptional gene expression regulation. Disorders of this process, particularly through RNA-binding proteins, is associated with the development and progression of a number of diseases, including cancers. However, the molecular mechanisms underlying the initiation of protein synthesis are intricate, making it difficult to find a drug that interferes with this process. Chemical probes are useful in elucidating the structures of RNA-protein complex and molecular mechanism of biological events. Moreover, some of these chemical probes show certain therapeutic benefits and can be further developed as leading compounds. Here, we will briefly review the general process and mechanism of protein synthesis, and emphasis on chemical probes in examples of probing the RNA structural changes and RNA-protein interactions. Moreover, the therapeutic potential of these probes is also discussed to give a comprehensive understanding.
Assuntos
Biologia Molecular/métodos , Biossíntese de Proteínas/efeitos dos fármacos , RNA/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Conformação de Ácido Nucleico/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA/química , RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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-quadruplex is a special secondary structure of nucleic acids in guanine-rich sequences of genome. G-quadruplexes have been proved to be involved in the regulation of replication, DNA damage repair, and transcription and translation of oncogenes or other cancer-related genes. Therefore, targeting G-quadruplexes has become a novel promising anti-tumor strategy. Different kinds of small molecules targeting the G-quadruplexes have been designed, synthesized, and identified as potential anti-tumor agents, including molecules directly bind to the G-quadruplex and molecules interfering with the binding between the G-quadruplex structures and related binding proteins. This review will explore the feasibility of G-quadruplex ligands acting as anti-tumor drugs, from basis to application. Meanwhile, since helicase is the most well-defined G-quadruplex-related protein, the most extensive research on the relationship between helicase and G-quadruplexes, and its meaning in drug design, is emphasized.
Assuntos
Desenvolvimento de Medicamentos , Quadruplex G , Ligantes , Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes.
Assuntos
Quadruplex G , Dobramento de RNA , RNA/análise , RNA/química , Sobrevivência Celular , Corantes Fluorescentes/química , Células HeLa , Humanos , Imagem ÓpticaRESUMO
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
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
c-MYC is an important oncogene that is considered as an effective target for anticancer therapy. Regulation of this gene's transcription is one avenue for c-MYC-targeting drug design. Direct binding to a transcription factor and generating the intervention of a transcriptional programme appears to be an effective way to modulate gene transcription. NM23-H2 is a transcription factor for c-MYC and is proven to be related to the secondary structures in the promoter. Here, we first screened our small-molecule library for NM23-H2 binders and then sifted through the inhibitors that could target and interfere with the interaction process between NM23-H2 and the guanine-rich promoter sequence of c-MYC. As a result, a quinazolone derivative, SYSU-ID-01: , showed a significant interference effect towards NM23-H2 binding to the guanine-rich promoter DNA sequence. Further analyses of the compound-protein interaction and the protein-DNA interaction provided insight into the mode of action for SYSU-ID-01: . Cellular evaluation results showed that SYSU-ID-01: could abrogate NM23-H2 binding to the c-MYC promoter, resulting in downregulation of c-MYC transcription and dramatically suppressed HeLa cell growth. These findings provide a new way of c-MYC transcriptional control through interfering with NM23-H2 binding to guanine-rich promoter sequences by small molecules.
Assuntos
Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Quinazolinonas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Apoptose , Células Cultivadas , DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Camundongos , Nucleosídeo NM23 Difosfato Quinases/química , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/biossíntese , Quinazolinonas/química , Bibliotecas de Moléculas PequenasRESUMO
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
BACKGROUND: Telomere is protected by its G-quadruplex, T-loop structure, telomerase, and binding protein complex. Protein POT1 (protection of telomeres 1) is one subunit of telomere binding protein complex Shelterin. POT1 acts as a regulator of telomerase-dependent telomere length, and it can help telomere to form D-loop structure to stabilize telomere. POT1 protects telomere ends from ATR-dependent DNA damage response as well. METHODS: Extensive methods were used, including CD, EMSA, ITC, PCR stop assay, luciferase reporter assay, quantitative real-time PCR, Western blot, chromatin immunoprecipitation (Ch-IP), cloning, expression and purification of proteins. RESULTS: We found a new G-rich 30-base-pair long sequence (P-pot1 G18) located from -165 to -136 base pairs upstream of the translation starting site of protein POT1. This sequence in the promoter region of pot1 gene formed G-quadruplex resulting in down-regulation of pot1 gene transcription. This G-rich sequence is close to a binding site "TCCC" for transcription factor hnRNP K (heterogeneous nuclear ribonucleoprotein K), and its conversion to G-quadruplex prevented the access of hnRNP K to this binding site. The binding of hnRNP K could up-regulate pot1 gene transcription. TMPyP4 (meso-tetra(N-methyl-4-pyridyl)porphine) has been widely used as G-quadruplex binding ligand, which stabilized the G-quadruplex in vitro and in cellulo, resulting in down-regulation of pot1 gene transcription. CONCLUSIONS: This G-quadruplex might become a potentially new drug target for antitumor agents. GENERAL SIGNIFICANCE: Our results first demonstrated that G-quadruplex formation can affect the binding of transcription factor to its nearby binding site, and thus making additional influence to gene transcription.
Assuntos
Quadruplex G/efeitos dos fármacos , Proteínas de Ligação a Telômeros/biossíntese , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Sítios de Ligação , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Porfirinas/farmacologia , Ligação Proteica , Complexo Shelterina , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/químicaRESUMO
BACKGROUND: Cellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases by controlling cell proliferation and survival to mediate neural crest expansion. CNBP has been found to bind single-stranded nucleic acid and promote rearrangements of nucleic acid secondary structure in an ATP-independent manner, acting as a nucleic acid chaperone. METHODS: A variety of methods were used, including cell viability assays, wound-scratch assays, chemotaxis assays, invasion assays, circular dichroic (CD) spectroscopy, NMR spectroscopy, chromatin immunoprecipitation, expression and purification of recombinant human CNBP, electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) analyses, luciferase reporter assay, Western blotting, and isothermal titration calorimetry (ITC). RESULTS: Up-regulation of CNBP induced human fibrosarcoma cell death and suppressed fibrosarcoma cell motility and invasiveness. It was found that CNBP transcriptionally down-regulated the expression of heterogeneous ribonucleoprotein K (hnRNP K) through its conversion of a G-rich sequence into G-quadruplex in the promoter of hnRNP K. G-quadruplex stabilizing ligand tetra-(N-methyl-4-pyridyl) porphyrin (TMPyP4) could interact with and stabilize the G-quadruplex, resulting in downregulation of hnRNP K transcription. CONCLUSIONS: CNBP overexpression caused increase of cell death and suppression of cell metastasis through its induction of G-quadruplex formation in the promoter of hnRNP K resulting in hnRNP K down-regulation. GENERAL SIGNIFICANCE: The present result provided a new solution for controlling hnRNP K expression, which should shed light on new anticancer drug design and development.
Assuntos
Fibrossarcoma/genética , Quadruplex G , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Proteínas de Ligação a RNA/genética , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , Fibrossarcoma/patologia , Transferência Ressonante de Energia de Fluorescência , Regulação Neoplásica da Expressão Gênica/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/biossíntese , Humanos , Metástase Neoplásica , Ácidos Nucleicos/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Angiogenesis is thought to be important in tumorigenesis and tumor progress. Vascular endothelial growth factor (VEGF) is a pluripotent cytokine and angiogenic growth factor that plays crucial roles in embryonic development and tumor progression. In many types of cancer, VEGF is overexpressed and is generally associated with tumor progression and survival rate. The polypurine/polypyrimidine sequence located upstream of the promoter region in the human VEGF gene can form specific parallel G-quadruplex structures, raising the possibility for transcriptional control of VEGF through G-quadruplex ligands. METHODS: PCR stop assay, circular dichroism (CD) spectra, RNA extraction and RT-PCR, enzyme-linked immunosorbent assay (ELISA), luciferase Assays, cell scrape test, xCELLigence real-time cell analysis (RTCA), and chick embryo chorioallantoic membrane (CAM) assay. RESULTS AND CONCLUSIONS: We found that quindoline derivatives can interact with the G-rich DNA sequences of the VEGF promoter to stabilize this G-quadruplex and suppress the transcription and expression of the VEGF protein. We also demonstrated that these derivatives exhibit potential anti-angiogenic activity in chick embryos and antitumor activity, including the inhibition of cell proliferation and migration. GENERAL SIGNIFICANCE: Our new findings have significances not only for understanding the mechanism of the G-quadruplex ligands mediating the VEGF transcription inhibition, but also for exploring a new anti-tumor strategy to blocking the transcription of VEGF to inhibit the angiogenesis in cancer cells.
Assuntos
Alcaloides , Ilhas de CpG , Indóis , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Regiões Promotoras Genéticas , Quinolinas , Transcrição Gênica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Alcaloides/química , Alcaloides/farmacocinética , Alcaloides/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Dicroísmo Circular , Humanos , Indóis/química , Indóis/farmacocinética , Indóis/farmacologia , Ligantes , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Quinolinas/química , Quinolinas/farmacocinética , Quinolinas/farmacologia , Transcrição Gênica/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Accelerated proliferation of solid tumor and hematologic cancer cells is related to accelerated transcription of ribosomal DNA by the RNA polymerase I to produce elevated level of ribosomal RNA. Therefore, down-regulation of RNA polymerase I transcription in cancer cells is an important anticancer therapeutic strategy. METHODS: A variety of methods were used, including cloning, expression and purification of protein, electrophoretic mobility shift assay (EMSA), circular dichroic (CD) spectroscopy, CD-melting, isothermal titration calorimetry (ITC), chromatin immunoprecipitation (Ch-IP), RNA interference, RT-PCR, Western blot, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell assay. RESULTS: Our results showed that 2,4-disubstituted quinazoline derivative Sysu12d could down-regulate c-myc through stabilization of c-myc promoter G-quadruplex, resulting in down-regulation of nucleolin expression. Sysu12d could also disrupt nucleolin/G-quadruplex complex. Both of the above contributed to the down-regulation of ribosomal RNA synthesis, followed by activation of p53 and then cancer cell apoptosis. CONCLUSIONS: These mechanistic studies set up the basis for further development of Sysu12d as a new type of lead compound for cancer treatment. GENERAL SIGNIFICANCE: 2,4-Disubstituted quinazoline derivatives may have multi-functional effect for cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinazolinas/farmacologia , Antineoplásicos/química , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Células HL-60 , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Quinazolinas/química , RNA Neoplásico/biossíntese , RNA Ribossômico/biossíntese , Proteína Supressora de Tumor p53/metabolismoRESUMO
G-quadruplexes are higher-order nucleic acid structures that have attracted extensive attention because of their biological significance and potential applications in supramolecular chemistry. An ever-increasing interest in G-quadruplexes has promoted the development of selective and sensitive fluorescent probes as research tools for these structures. However, most current studies primarily focus on the improved selectivity of probes for G-quadruplexes. Their detection limits or ways to improve their detection limits are rarely described. In this study, a new set of di-substituted triarylimidazole fluorescent probes were designed and synthesized, with the aim of upgrading the detection limit of a lead triarylimidazole IZCM-1 for G-quadruplexes. Among these compounds, IZCM-7 was the most promising candidate. The limit of detection (LOD) value of IZCM-7 for the G-quadruplex was up to 3 nM in solution and up to 5 ng in a gel matrix. These values were significantly improved in comparison with those of IZCM-1. Further biophysical studies revealed that the fluorescence quantum yield and binding affinity of IZCM-7 for G-quadruplexes were markedly increased, and these two factors might be responsible for the significantly improved detection limit of IZCM-7. In addition, the sensitive and selective fluorescence performance of IZCM-7 for G-quadruplexes remained the same even in the presence of large amounts of non-G-quadruplex competitors, suggesting its promising application prospect.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes/química , Quadruplex G , Animais , Sequência de Bases , Bovinos , DNA/química , DNA/genética , Corantes Fluorescentes/síntese química , Imidazóis/síntese química , Imidazóis/química , Limite de Detecção , Simulação de Dinâmica Molecular , Fenômenos Ópticos , Espectrometria de FluorescênciaRESUMO
Bouchardatine (1), a naturally occurring ß-indoloquinazoline alkaloid, was synthesized. For the first time, the lipid-lowering effect and mechanism of 1 was investigated in 3T3-L1 adipocytes. Our study showed that 1 could significantly reduce lipid accumulation without cytotoxicity and mainly inhibited early differentiation of adipocyte through proliferation inhibition and cell cycle arrested in dose-dependent manner. Furthermore, the inhibition of early differentiation was reflected by down-regulation of key regulators of adipogenesis/lipogenesis, including CCAAT enhancer binding proteins (C/EBPß, C/EBPδ, C/EBPα), peroxisome proliferator-activated receptors γ (PPARγ) and sterol-regulatory element binding protein-1c (SREBP-1c), in both of mRNA and protein levels. Subsequently decreasing the protein levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), and stearyl coenzyme A desaturated enzyme 1 (SCD-1), the rate-limited metabolic enzymes of fatty acid synthesis, were also observed. Further studies revealed that 1 persistently activated adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) during differentiation, suggesting that the AMPK may be an upstream mechanism for the effect of 1 on adipogenesis and lipogenesis. Our data suggest that 1 can be a candidate for the development of new therapeutic drugs against obesity and related metabolic disorders.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Alcaloides Indólicos/química , Lipogênese/efeitos dos fármacos , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Alcaloides Indólicos/farmacologia , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
BACKGROUND: G-quadruplexes are promising therapeutic targets for small molecules. In general, the introduction of steady positive charges through the in situ alkylation of nitrogen atoms within potential G-quadruplex ligands can significantly improve their quadruplex binding and stabilization abilities. However, our previous studies on bisaryldiketene derivatives showed that the derivative M4, whose central piperidone moiety is quaternized, exhibits a poor G-quadruplex stabilization ability. METHODS: To clarify this unusual finding, CD, ITC, UV and NMR analyses were performed to determine the binding behaviors of M4 and its non-quaternized analog M2 to G-quadruplex DNA [d(TGGGT)]4. Molecular modeling approaches were also employed to help illustrate ligand-quadruplex DNA interactions. RESULTS: The CD melting and ITC analyses revealed that M2 exhibited much stronger stabilization and binding abilities to [d(TGGGT)]4 compared to M4. Moreover, the CD and ITC analyses in combination with UV, NMR and MD simulations revealed that M2 tended to be end-stacked on the G-quartet, whereas M4 tended to be bound in the groove region. Analysis of the electrostatic potential showed that the charged surface of M4 was more positive than that of M2 and other reported ligands that bind to the G-quadruplex via end-stacking interactions. CONCLUSIONS: The results indicated that the different positively charged surfaces of M2 and M4 might be the key reason for their different binding modes. These different binding modes also lead to different binding affinities and stabilization abilities for [d(TGGGT)]4. GENERAL SIGNIFICANCE: These results provide new clues for the rational design of G-quadruplex-binding small molecules with steady positive charges.
Assuntos
Quadruplex G , Lactonas/química , Bibliotecas de Moléculas Pequenas/química , Calorimetria/métodos , Dicroísmo Circular/métodos , DNA/química , Cinética , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Simulação de Dinâmica Molecular , Espectrofotometria Ultravioleta/métodos , TermodinâmicaRESUMO
BACKGROUND: Guanine-rich sequence of c-myc nuclease hypersensitive element (NHE) III1 is known to fold in G-quadruplex and subsequently serves as a transcriptional silencer. Cellular nucleic-acid-binding protein (CNBP), a highly conserved zinc-finger protein with multiple biological functions, could bind to c-myc NHE III1 region, specifically to the single strand G-rich sequence. METHODS: In the present study, a variety of methods, including cloning, expression and purification of protein, EMSA, CD, FRET, Ch-IP, RNA interference, luciferase reporter assay, SPR, co-immunoprecipitation, and co-transfection, were applied to investigate the mechanism for the role of CNBP in regulating c-myc transcription. RESULTS: We found that human CNBP specifically bound to the G-rich sequence of c-myc NHE III1 region both in vitro and in cellulo, and subsequently promoted the formation of G-quadruplex. CNBP could induce a transient decrease followed by an increase in c-myc transcription in vivo. The interaction of CNBP with NM23-H2 was responsible for the increase of c-myc transcription. CONCLUSIONS: Based on above experimental results, a new mechanism, involving G-quadruplex related CNBP/NM23-H2 interaction, for the regulation of c-myc transcription was proposed. GENERAL SIGNIFICANCE: These findings indicated that the regulation of c-myc transcription through NHE III1 region might be governed by mechanisms involving complex protein-protein interactions, and suggested a new possibility of CNBP as a potential anti-cancer target based on CNBP's biological function in c-myc transcription.
Assuntos
Genes myc , Proteínas de Ligação a RNA/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Imunoprecipitação da Cromatina , Dicroísmo Circular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Transferência Ressonante de Energia de Fluorescência , Humanos , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Ativação TranscricionalRESUMO
NRAS mutation is the second most common oncogenic factor in cutaneous melanoma. Inhibiting NRAS translation by stabilizing the G-quadruplex (G4) structure with small molecules seems to be a potential strategy for cancer therapy due to the NRAS protein's lack of a druggable pocket. To enhance the effects of previously reported G4 stabilizers quindoline derivatives, we designed and synthesized a novel series of quindoline derivatives with fork-shaped side chains by introducing (alkylamino)alkoxy side chains. Panels of experimental results showed that introducing a fork-shaped (alkylamino)alkoxy side chain could enhance the stabilizing abilities of the ligands against NRAS RNA G-quadruplexes and their anti-melanoma activities. One of them, 10b, exhibited good antitumor activity in the NRAS-mutant melanoma xenograft mouse model, showing the therapeutic potential of this kind of compounds.
Assuntos
Antineoplásicos , Desenho de Fármacos , Quadruplex G , GTP Fosfo-Hidrolases , Proteínas de Membrana , Quadruplex G/efeitos dos fármacos , Humanos , Animais , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Relação Estrutura-Atividade , Estrutura Molecular , Melanoma/tratamento farmacológico , Melanoma/patologia , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Indóis/química , Indóis/síntese química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , RNA/metabolismo , RNA/química , Biossíntese de Proteínas/efeitos dos fármacos , Alcaloides , QuinolinasRESUMO
The C-5-methylation of cytosine in the CpG islands is an important pattern for epigenetic modification of gene, which plays a key role in regulating gene transcription. G-quadruplex is an unusual DNA secondary structure formed in G-rich regions and is identified as a transcription repressor in some oncogenes, such as c-myc and bcl-2. In the present study, the results from CD spectrum and FRET assay showed that the methylation of cytosine in the CpG islands could induce a conformational change of the G-quadruplex in the P1 promoter of bcl-2, and greatly increase the thermal-stability of this DNA oligomer. Moreover, the methylation of cytosine in the G-quadruplex could protect the structure from the disruption by the complementary strand, showing with the increasing ability to arrest the polymerase in PCR stop assay. This data indicated that the stabilization of the G-quadruplex structure in the CpG islands might be involved in the epigenetical transcriptional regulation for specific genes through the C-5-methylation modification pattern.