RESUMEN
Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of -1 PRF in SARS-CoV-2 also inhibited -1 PRF in a range of bat CoVs-the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed -1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.
Asunto(s)
Antivirales/farmacología , Coronavirus/efectos de los fármacos , Coronavirus/genética , Mutación del Sistema de Lectura , Sistema de Lectura Ribosómico/efectos de los fármacos , Proteínas Virales/antagonistas & inhibidores , Animales , Antivirales/uso terapéutico , Quirópteros/virología , Coronavirus/clasificación , Infecciones por Coronavirus/tratamiento farmacológico , Conformación de Ácido Nucleico , ARN Mensajero/genética , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
The ongoing COVID-19 pandemic, periodic recurrence of viral infections, and the emergence of challenging variants has created an urgent need of alternative therapeutic approaches to combat the spread of viral infections, failing to which may pose a greater risk to mankind in future. Resilience against antiviral drugs or fast evolutionary rate of viruses is stressing the scientific community to identify new therapeutic approaches for timely control of disease. Host metabolic pathways are exquisite reservoir of energy to viruses and contribute a diverse array of functions for successful replication and pathogenesis of virus. Targeting the host factors rather than viral enzymes to cease viral infection, has emerged as an alternative antiviral strategy. This approach offers advantage in terms of increased threshold to viral resistance and can provide broad-spectrum antiviral action against different viruses. The article here provides substantial review of literature illuminating the host factors and molecular mechanisms involved in innate/adaptive responses to viral infection, hijacking of signalling pathways by viruses and the intracellular metabolic pathways required for viral replication. Host-targeted drugs acting on the pathways usurped by viruses are also addressed in this study. Host-directed antiviral therapeutics might prove to be a rewarding approach in controlling the unprecedented spread of viral infection, however the probability of cellular side effects or cytotoxicity on host cell should not be ignored at the time of clinical investigations.
Asunto(s)
Antivirales/farmacología , Virus ARN Monocatenarios Positivos/efectos de los fármacos , Animales , Citocinas/metabolismo , Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/fisiología , Glicosilación/efectos de los fármacos , Humanos , Inmunidad/efectos de los fármacos , Inmunidad/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/fisiología , Poliaminas/metabolismo , Virus ARN Monocatenarios Positivos/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ubiquitinación/efectos de los fármacos , Ubiquitinación/fisiologíaRESUMEN
Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (-1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro -1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential -1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.
Asunto(s)
Antivirales/farmacología , Sistema de Lectura Ribosómico/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Quinolinas/farmacología , SARS-CoV-2/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Células A549 , Animales , Línea Celular , Sistema de Lectura Ribosómico/fisiología , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Bibliotecas de Moléculas Pequeñas , Zoonosis Virales/virología , Replicación Viral/efectos de los fármacosRESUMEN
Treatment of HIV-1 has largely involved targeting viral enzymes using a cocktail of inhibitors. However, resistance to these inhibitors and toxicity in the long term have pushed the field to identify new therapeutic targets. To that end, -1 programmed ribosomal frameshifting (-1 PRF) has gained attention as a potential node for therapeutic intervention. In this process, a ribosome moves one nucleotide backward in the course of translating a mRNA, revealing a new reading frame for protein synthesis. In HIV-1, -1 PRF allows the virus to regulate the ratios of enzymatic and structural proteins as needed for correct viral particle assembly. Two RNA structural elements are central to -1 PRF in HIV: a slippery sequence and a highly conserved stable hairpin called the HIV-1 frameshifting stimulatory signal (FSS). Dysregulation of -1 PRF is deleterious for the virus. Thus, -1 PRF is an attractive target for new antiviral development. It is important to note that HIV-1 is not the only virus exploiting -1 PRF for regulating production of its proteins. Coronaviruses, including the COVID-19 pandemic virus SARS-CoV-2, also rely on -1 PRF. In SARS-CoV-2 and other coronaviruses, -1 PRF is required for synthesis of RNA-dependent RNA polymerase and several other nonstructural proteins. Coronaviruses employ a more complex RNA structural element for regulating -1 PRF called a pseudoknot. The purpose of this Account is primarily to review the development of molecules targeting HIV-1 -1 PRF. These approaches are case studies illustrating how the entire pipeline from screening to the generation of high-affinity leads might be implemented. We consider both target-based and function-based screening, with a particular focus on our group's approach beginning with a resin-bound dynamic combinatorial library (RBDCL) screen. We then used rational design approaches to optimize binding affinity, selectivity, and cellular bioavailability. Our tactic is, to the best of our knowledge, the only study resulting in compounds that bind specifically to the HIV-1 FSS RNA and reduce infectivity of laboratory and drug-resistant strains of HIV-1 in human cells. Lessons learned from strategies targeting -1 PRF HIV-1 might provide solutions in the development of antivirals in areas of unmet medical need. This includes the development of new frameshift-altering therapies for SARS-CoV-2, approaches to which are very recently beginning to appear.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Antivirales/química , Técnicas Químicas Combinatorias , Sistema de Lectura Ribosómico/efectos de los fármacos , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed -1 ribosomal frameshift (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other betacoronaviruses. Consistent with the essential role of -1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting -1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.
Asunto(s)
Antivirales/farmacología , Sistema de Lectura Ribosómico/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Betacoronavirus , Chlorocebus aethiops , Fluoroquinolonas/farmacología , Sistema de Lectura Ribosómico/genética , Mutación , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , SARS-CoV-2/fisiología , Células VeroRESUMEN
Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.
Asunto(s)
Antivirales/farmacología , Antivirales/uso terapéutico , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/genética , Virosis/tratamiento farmacológico , Virosis/virología , Animales , Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/genética , Regulación Viral de la Expresión Génica/genética , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Humanos , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genéticaRESUMEN
Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.
Asunto(s)
Sistema de Lectura Ribosómico , ARN Viral/genética , Ribosomas/ultraestructura , SARS-CoV-2/genética , Proteínas Virales/biosíntesis , Animales , Antivirales/farmacología , Codón de Terminación , ARN Polimerasa Dependiente de ARN de Coronavirus/biosíntesis , ARN Polimerasa Dependiente de ARN de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Microscopía por Crioelectrón , Fluoroquinolonas/farmacología , Sistema de Lectura Ribosómico/efectos de los fármacos , Genoma Viral , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos Moleculares , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Pliegue de Proteína , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/química , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.
Asunto(s)
Antivirales/farmacología , Coronavirus/efectos de los fármacos , Coronavirus/genética , Sistema de Lectura Ribosómico/efectos de los fármacos , Antivirales/química , Antivirales/uso terapéutico , Coronavirus/crecimiento & desarrollo , Coronavirus/fisiología , Infecciones por Coronavirus/tratamiento farmacológico , Sistema de Lectura Ribosómico/genética , Sistema de Lectura Ribosómico/fisiología , Regulación Viral de la Expresión Génica , Humanos , Mutación , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/fisiología , Replicación ViralRESUMEN
Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.
Asunto(s)
Presentación de Antígeno , Mutación del Sistema de Lectura , Melanoma/inmunología , Péptidos/genética , Péptidos/inmunología , Biosíntesis de Proteínas/inmunología , Linfocitos T/inmunología , Línea Celular , Codón/genética , Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/genética , Sistema de Lectura Ribosómico/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/inmunología , Interferón gamma/farmacología , Melanoma/patología , Péptidos/química , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteoma , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Triptófano/deficiencia , Triptófano/genética , Triptófano/metabolismoRESUMEN
SARS-CoV-2 uses -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins. Because modulating -1 PRF can attenuate the virus, ligands binding to the RNA pseudoknot that stimulates -1 PRF may have therapeutic potential. Mutations in the pseudoknot have occurred during the pandemic, but how they affect -1 PRF efficiency and ligand activity is unknown. Studying a panel of six mutations in key regions of the pseudoknot, we found that most did not change -1 PRF levels, even when base-pairing was disrupted, but one led to a striking 3-fold decrease, suggesting SARS-CoV-2 may be less sensitive to -1 PRF modulation than expected. Examining the effects of a small-molecule -1 PRF inhibitor active against SARS-CoV-2, it had a similar effect on all mutants tested, regardless of basal -1 PRF efficiency, indicating that anti-frameshifting activity can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of -1 PRF.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Sistema de Lectura Ribosómico/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Antivirales/química , Betacoronavirus/genética , COVID-19 , Infecciones por Coronavirus/virología , Humanos , Ligandos , Mutación/efectos de los fármacos , Pandemias , Neumonía Viral/virología , ARN Mensajero/genética , ARN Viral/genética , SARS-CoV-2 , Bibliotecas de Moléculas Pequeñas/química , Proteínas Virales/genéticaRESUMEN
Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.
Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/tratamiento farmacológico , Diseño de Fármacos , Sistema de Lectura Ribosómico/efectos de los fármacos , Neumonía Viral/tratamiento farmacológico , ARN Viral/genética , Betacoronavirus/química , COVID-19 , Regulación Viral de la Expresión Génica , Humanos , Pandemias , ARN Viral/química , SARS-CoV-2 , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
In a recent paper by Dvela-Levitt et al., chemical screening using an immunofluorescent assay identified a compound that caused removal of a dominant-inherited misfolded secretory protein, mucin1-frameshifted, from an intracellular location in immortalized renal epithelial cells of a patient affected with progressive medullary cystic kidney disease. This illustrates the power of chemical screening at the cellular level to address specific proteinopathies and the utility of such compounds to illuminate novel cellular pathways that can clear toxic proteins.
Asunto(s)
Sistema de Lectura Ribosómico , Enfermedades Renales Quísticas/metabolismo , Mucina-1/química , Animales , Evaluación Preclínica de Medicamentos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Sistema de Lectura Ribosómico/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Enfermedades Renales Quísticas/tratamiento farmacológico , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/fisiopatología , Ratones , Mucina-1/genética , Mucina-1/metabolismo , Pliegue de Proteína/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Minus-one programmed ribosomal frameshifting (-1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2-8 nucleotides downstream from the slippery site. Upon -1 RF, the ribosomal reading frame is shifted from the normal zero frame to the -1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically modified peptide nucleic acid (PNA) L and Q monomers to recognize G-C and C-G Watson-Crick base pairs, respectively, through major-groove parallel PNA·RNA-RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free in vitro translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically modified PNAs targeting model mRNA hairpins stimulate -1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs.
Asunto(s)
Sistema de Lectura Ribosómico/efectos de los fármacos , Ácidos Nucleicos de Péptidos/farmacología , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Sistema Libre de Células , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , Biosíntesis de Proteínas , ConejosRESUMEN
The HIV-1 frameshift-stimulating (FSS) RNA, a regulatory RNA of critical importance in the virus' life cycle, has been posited as a novel target for anti-HIV drug development. We report the synthesis and evaluation of triazole-containing compounds able to bind the FSS with high affinity and selectivity. Readily accessible synthetically, these compounds are less toxic than previously reported olefin congeners. We show for the first time that FSS-targeting compounds have antiviral activity against replication-competent HIV in human cells, including a highly cytopathic, multidrug-resistant strain. These results support the viability of the HIV-1 FSS RNA as a therapeutic target and more generally highlight opportunities for synthetic molecule-mediated interference with protein recoding in a wide range of organisms.
Asunto(s)
Sistema de Lectura Ribosómico/efectos de los fármacos , VIH-1/genética , ARN Viral/efectos de los fármacos , Triazoles/farmacología , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular , VIH-1/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , ARN Viral/genéticaRESUMEN
Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate -1 programmed ribosomal frameshifting (PRF), suggesting -1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the -1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent -1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of -1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent -1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA-ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific -1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application.
Asunto(s)
Sistema de Lectura Ribosómico , Ligandos , ARN Viral/química , Riboswitch , Animales , Secuencia de Bases , Línea Celular , Sistema de Lectura Ribosómico/efectos de los fármacos , Humanos , Secuencias Invertidas Repetidas , Mamíferos , Modelos Biológicos , Conformación de Ácido Nucleico , ARN Viral/genética , Riboswitch/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Biología Sintética , Teofilina/química , Teofilina/farmacologíaRESUMEN
In this issue of Molecular Cell, Gupta et al. (2013a) describe a novel, antibiotic-dependent ribosomal frameshifting event that activates translation of an antibiotic resistance gene.
Asunto(s)
Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/genética , Regulación de la Expresión Génica/efectos de los fármacos , Macrólidos/farmacología , Ribosomas/efectos de los fármacos , Ribosomas/genéticaAsunto(s)
Antibacterianos/farmacología , Sistema de Lectura Ribosómico/efectos de los fármacos , Cetólidos/farmacología , Regiones no Traducidas 5'/genética , Farmacorresistencia Bacteriana/genética , Sistema de Lectura Ribosómico/genética , Sistemas de Lectura Abierta/genética , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Ribosomas/efectos de los fármacos , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The expression of many genes is controlled by upstream ORFs (uORFs). Typically, the progression of the ribosome through a regulatory uORF, which depends on the physiological state of the cell, influences the expression of the downstream gene. In the classic mechanism of induction of macrolide resistance genes, antibiotics promote translation arrest within the uORF, and the static ribosome induces a conformational change in mRNA, resulting in the activation of translation of the resistance cistron. We show that ketolide antibiotics, which do not induce ribosome stalling at the uORF of the ermC resistance gene, trigger its expression via a unique mechanism. Ketolides promote frameshifting at the uORF, allowing the translating ribosome to invade the intergenic spacer. The dynamic unfolding of the mRNA structure leads to the activation of resistance. Conceptually similar mechanisms may control other cellular genes. The identified property of ketolides to reduce the fidelity of reading frame maintenance may have medical implications.
Asunto(s)
Sistema de Lectura Ribosómico/efectos de los fármacos , Sistema de Lectura Ribosómico/genética , Regulación de la Expresión Génica/efectos de los fármacos , Macrólidos/farmacología , Ribosomas/efectos de los fármacos , Ribosomas/genética , Secuencia de Aminoácidos , Antibacterianos/farmacología , Secuencia de Bases , Cetólidos/farmacología , Metiltransferasas/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/efectos de los fármacos , Biosíntesis de Proteínas , ARN Mensajero/genéticaRESUMEN
2-Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. The bulky N-acetylated dG-AAF lesion can induce various frameshift mutations depending on the base sequence around the lesion. We hypothesized that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. The objective of the present study was to probe the structural/conformational basis of various dG-AAF-induced SMIs formed during translesion synthesis. We performed spectroscopic, thermodynamic, and molecular dynamics studies of several AAF-modified 16-mer model DNA duplexes, including fully paired and -1, -2, and -3 deletion duplexes of the 5'-CTCTCGATG[FAAF]CCATCAC-3' sequence and an additional -1 deletion duplex of the 5'-CTCTCGGCG[FAAF]CCATCAC-3' NarI sequence. Modified deletion duplexes existed in a mixture of external B and stacked S conformers, with the population of the S conformer being 'GC'-1 (73%) > 'AT'-1 (72%) > full (60%) > -2 (55%) > -3 (37%). Thermodynamic stability was in the order of -1 deletion > -2 deletion > fully paired > -3 deletion duplexes. These results indicate that the stacked S-type conformer of SMIs is thermodynamically more stable than the conformationally flexible external B conformer. Results from the molecular dynamics simulations indicate that perturbation of base stacking dominates the relative stability along with contributions from bending, duplex dynamics, and solvation effects that are important in specific cases. Taken together, these results support a hypothesis that the conformational and thermodynamic stabilities of the SMIs are critical determinants for the induction of frameshift mutations.
Asunto(s)
2-Acetilaminofluoreno/farmacología , ADN/efectos de los fármacos , Mutación del Sistema de Lectura/genética , Sistema de Lectura Ribosómico/efectos de los fármacos , Mutagénesis/efectos de los fármacos , Termodinámica , 2-Acetilaminofluoreno/química , ADN/genética , Modelos Químicos , Simulación de Dinámica Molecular , Estructura MolecularRESUMEN
Programmed -1 translational frameshifting is an essential event in the replication cycle of HIV. Frameshifting is required for expression of the viral Pol proteins, and drug-like molecules that target this process may inhibit HIV replication. A small molecule stimulator of HIV-1 frameshifting and inhibitor of viral replication, DB213 (RG501), was previously discovered from a high-throughput screen. However, the mechanistic basis for this compound's effects was unknown, and to date no structural information exists for small molecule effectors of frameshifting. Here, we investigate the binding of DB213 to the frameshift site RNA and have determined the structure of this complex by NMR. Binding of DB213 stabilizes the RNA and increases its melting temperature by 10 °C. The ligand binds to a primary site on the RNA stem-loop, although nonspecific interactions are also detected. The compound binds in the major groove and spans a distance of 9 base pairs. DB213 hydrogen bonds to phosphate groups on opposite sides of the major groove and alters the conformation of a conserved GGA bulge in the RNA. This study may provide a starting point for structure-based optimization of compounds targeting the HIV-1 frameshift site RNA.