RESUMEN
CST (CTC1-STN1-TEN1) is a telomere associated complex that binds ssDNA and is required for multiple steps in telomere replication, including termination of G-strand extension by telomerase and synthesis of the complementary C-strand. CST contains seven OB-folds which appear to mediate CST function by modulating CST binding to ssDNA and the ability of CST to recruit or engage partner proteins. However, the mechanism whereby CST achieves its various functions remains unclear. To address the mechanism, we generated a series of CTC1 mutants and studied their effect on CST binding to ssDNA and their ability to rescue CST function in CTC1-/- cells. We identified the OB-B domain as a key determinant of telomerase termination but not C-strand synthesis. CTC1-ΔB expression rescued C-strand fill-in, prevented telomeric DNA damage signaling and growth arrest. However, it caused progressive telomere elongation and the accumulation of telomerase at telomeres, indicating an inability to limit telomerase action. The CTC1-ΔB mutation greatly reduced CST-TPP1 interaction but only modestly affected ssDNA binding. OB-B point mutations also weakened TPP1 association, with the deficiency in TPP1 interaction tracking with an inability to limit telomerase action. Overall, our results indicate that CTC1-TPP1 interaction plays a key role in telomerase termination.
Asunto(s)
Telomerasa , Humanos , Línea Celular , ADN de Cadena Simple/genética , Mutación , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
The c-myc oncogene is an important regulator for cell growth and differentiation, and its aberrant overexpression is closely related to the occurrence and development of various cancers. Thus, the suppression of c-myc transcription and expression has been investigated for cancer treatment. In this study, various new bisacridine derivatives were synthesized and evaluated for their binding with c-myc promoter G-quadruplex and i-motif. We found that a9 could bind to and stabilize both G-quadruplex and i-motif, resulting in the downregulation of c-myc gene transcription. a9 could inhibit cancer cell proliferation and induce SiHa cell apoptosis and cycle arrest. a9 exhibited tumor growth inhibition activity in a SiHa xenograft tumor model, which might be related to its binding with c-myc promoter G-quadruplex and i-motif. Our results suggested that a9 as a dual G-quadruplex/i-motif binder could be effective in both oncogene replication and transcription and become a promising lead compound for further development with improved potency and selectivity.
Asunto(s)
Acridinas/química , Antineoplásicos/síntesis química , G-Cuádruplex , Proteínas Proto-Oncogénicas c-myc/genética , Acridinas/metabolismo , Acridinas/farmacología , Acridinas/uso terapéutico , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Sitios de Unión , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Aberrant overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key feature in oncogenesis and progression of many human cancers. hnRNP K has been found to be a transcriptional activator to up-regulate c-myc gene transcription, a critical proto-oncogene for regulation of cell growth and differentiation. Therefore, down-regulation of c-myc transcription by inhibiting hnRNP K through disrupting its binding to c-myc gene promoter is a potential approach for cancer therapy. In the present study, we synthesized and screened a series of Quinoline derivatives and evaluated their binding affinity for hnRNP K. Among these derivatives, (E)-1-(4-methoxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (compound 25) was determined to be the first-reported hnRNP K binding ligand with its KD values of 4.6 and 2.6⯵M measured with SPR and MST, respectively. Subsequent evaluation showed that the binding of compound 25 to hnRNP K could disrupt its unfolding of c-myc promoter i-motif, resulting in down-regulation of c-myc transcription. Compound 25 showed a selective anti-proliferative effect on human cancer cell lines with IC50 values ranged from 1.36 to 3.59⯵M. Compound 25 exhibited good tumor growth inhibition in a Hela xenograft tumor model, which might be related to its binding with hnRNP K. These findings illustrated that inhibition of DNA-binding protein hnRNP K by compound 25 could be a new and selective strategy of regulating oncogene transcription instead of targeting promoter DNA secondary structures such as G-quadruplexes or i-motifs.
Asunto(s)
Antineoplásicos/uso terapéutico , Ribonucleoproteína Heterogénea-Nuclear Grupo K/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Quinolinas/uso terapéutico , Transcripción Genética/efectos de los fármacos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Unión Proteica , Proto-Oncogenes Mas , Quinolinas/síntesis química , Quinolinas/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The human proto-oncogene neuroblastoma RAS ( NRAS) contains a guanine-rich sequence in the 5'-untranslated regions (5'-UTR) of the mRNA that could form an RNA G-quadruplex structure. This structure acts as a repressor for NRAS translation and could be a potential target for anticancer drugs. Our previous studies found an effective scaffold, the quindoline scaffold, for binding and stabilizing the DNA G-quadruplex structures. Here, on the basis of the previous studies and reported RNA-specific probes, a series of novel p-(methylthio)styryl substituted quindoline (MSQ) derivatives were designed, synthesized, and evaluated as NRAS RNA G-quadruplex ligands. Panels of experiments turned out that the introduction of p-(methylthio)styryl side chain could enhance the specific binding to the NRAS RNA G-quadruplex. One of the hits, 4a-10, showed strong stabilizing activity on the G-quadruplex and subsequently repressed NRAS's translation and inhibited tumor cells proliferation. Our finding provided a novel strategy to discover novel NRAS repressors by specifically binding to the RNA G-quadruplex in the 5'-UTR of mRNA.
Asunto(s)
Alcaloides/síntesis química , Alcaloides/farmacología , Diseño de Fármacos , G-Cuádruplex/efectos de los fármacos , GTP Fosfohidrolasas/genética , Indoles/síntesis química , Indoles/farmacología , Proteínas de la Membrana/genética , Quinolinas/síntesis química , Quinolinas/farmacología , ARN/química , Estireno/química , Alcaloides/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Química Sintética , Humanos , Indoles/química , Proto-Oncogenes Mas , Quinolinas/químicaRESUMEN
We synthesized a series of acridone derivatives for specific binding ligands of i-motifs. Subsequent evaluations showed that B19 could selectively bind to and stabilize the c-myc promoter i-motif without significant binding to the G-quadruplex and duplex DNA. This caused down-regulation of c-myc transcription and expression, resulting in tumor cell apoptosis.
Asunto(s)
Acridonas/farmacología , Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética/efectos de los fármacos , Acridonas/síntesis química , Acridonas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Ensayos de Selección de Medicamentos Antitumorales , Transferencia Resonante de Energía de Fluorescencia , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Estructura Molecular , Relación Estructura-Actividad , Transcripción Genética/genéticaRESUMEN
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.
Asunto(s)
Alcaloides/farmacología , Daño del ADN , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Quinolinas/farmacología , ARN Largo no Codificante/metabolismo , Telómero , Proteína 2 de Unión a Repeticiones Teloméricas/antagonistas & inhibidores , Alcaloides/metabolismo , Regulación Alostérica , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , G-Cuádruplex , Humanos , Indoles/metabolismo , Neoplasias/genética , Neoplasias/patología , Quinolinas/metabolismoRESUMEN
Angiogenesis is important in tumorigenesis and tumor progression. Human vascular endothelial growth factor (hVEGF) is an angiogenic growth factor that plays a crucial role in tumor progression. The G-rich region within the 5'-untranslated regions (5'-UTR) of hVEGF-A mRNA can form a "switchable" RNA G-quadruplex structure that is essential for a cap-independent translation initiation. We screened our small-molecule library for binders of this G-tract. One novel quinazoline derivative, compound 1, showed a significant specific interaction with the G-tract and destabilized the G-quadruplex structure. The results of cellular experiments revealed that compound 1 down-regulated hVEGF-A translation and significantly impeded tumor cells migration. We also found that compound 1 exhibited tumor-inhibiting activity in MCF-7 xenograft tumors, which might be related to its ability to reduce hVEGF expression. These findings present a new strategy of hVEGF-A translational control in which small molecules interact with G-quadruplex structure in the 5'UTR.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , G-Cuádruplex/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Quinolonas/farmacología , Triazoles/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Química Clic , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Quinolonas/síntesis química , Quinolonas/química , Relación Estructura-Actividad , Termodinámica , Triazoles/químicaRESUMEN
A series of 4-anilinoquinazoline derivatives were designed and synthesized as novel c-myc promoter G-quadruplex binding ligands. Subsequent biophysical and biochemical evaluation demonstrated that the introduction of aniline group at 4-position of quinazoline ring and two side chains with terminal amino group improved their binding affinity and stabilizing ability to G-quadruplex DNA. RT-PCR assay and Western blot showed that compound 7a could down-regulate transcription and expression of c-myc gene in Hela cells, which was consistent with the behavior of an effective G-quadruplex ligand targeting c-myc oncogene. More importantly, RTCA and colony formation assays indicated that 7a obviously inhibited Hela cells proliferation, without influence on normal primary cultured mouse mesangial cells. Flow cytometric assays suggested that 7a induced Hela cells to arrest in G0/G1 phase both in a time-dependent and dose-dependent manner.