RESUMO
BACKGROUND: Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. METHODS: SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19. RESULTS: Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 µM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. CONCLUSIONS: Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.
Assuntos
COVID-19 , Preparações Farmacêuticas , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Carbamatos , Chlorocebus aethiops , Humanos , Imidazóis , Pirrolidinas , RNA Viral , SARS-CoV-2 , Sofosbuvir/farmacologia , Valina/análogos & derivados , Células VeroRESUMO
With the recent global spread of new SARS-CoV-2 variants, there remains an urgent need to develop effective and variant-resistant oral drugs. Recently, we reported in vitro results validating the use of combination drugs targeting both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) as potential COVID-19 therapeutics. For the nucleotide analogues to be efficient SARS-CoV-2 inhibitors, two properties are required: efficient incorporation by RdRp and substantial resistance to excision by ExoN. Here, we have selected and evaluated nucleotide analogues with a variety of structural features for resistance to ExoN removal when they are attached at the 3' RNA terminus. We found that dideoxynucleotides and other nucleotides lacking both 2'- and 3'-OH groups were most resistant to ExoN excision, whereas those possessing both 2'- and 3'-OH groups were efficiently removed. We also found that the 3'-OH group in the nucleotide analogues was more critical than the 2'-OH for excision by ExoN. Since the functionally important sequences in Nsp14/10 are highly conserved among all SARS-CoV-2 variants, these identified structural features of nucleotide analogues offer invaluable insights for designing effective RdRp inhibitors that can be simultaneously efficiently incorporated by the RdRp and substantially resist ExoN excision. Such newly developed RdRp terminators would be good candidates to evaluate their ability to inhibit SARS-CoV-2 in cell culture and animal models, perhaps combined with additional exonuclease inhibitors to increase their overall effectiveness.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Antivirais/uso terapêutico , Exonucleases , Nucleotídeos/química , RNA Viral/genéticaRESUMO
SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Exonucleases/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anilidas/farmacologia , Animais , Sequência de Bases , Benzimidazóis/farmacologia , COVID-19/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Sinergismo Farmacológico , Exonucleases/genética , Exonucleases/metabolismo , Humanos , Prolina/farmacologia , Pirrolidinas/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Valina/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC50) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only Mpro, but also the 3'-5' exonuclease (ExoN).
Assuntos
Tratamento Farmacológico da COVID-19 , Flavonas , Isoflavonas , Flavonas/farmacologia , Flavonoides/farmacologia , Flavonóis/farmacologia , Humanos , Isoflavonas/farmacologia , Quempferóis , Simulação de Acoplamento Molecular , Inibidores de Proteases , Quercetina/farmacologia , SARS-CoV-2RESUMO
SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir's apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.
Assuntos
Tratamento Farmacológico da COVID-19 , Hepatite C , Humanos , SARS-CoV-2 , Antivirais/uso terapêutico , Sofosbuvir/farmacologia , Nucleosídeos/farmacologia , Monofosfato de Adenosina , Alanina , Hepacivirus , Hepatite C/tratamento farmacológico , PulmãoRESUMO
Mass spectrometry (MS)-based sequencing has advantages in direct sequencing of RNA, compared to cDNA-based RNA sequencing methods, as it is completely independent of enzymes and base complementarity errors in sample preparation. In addition, it allows for sequencing of different RNA modifications in a single study, rather than just one specific modification type per study. However, many technical challenges remain in de novo MS sequencing of RNA, making it difficult to MS sequence mixed RNAs or to differentiate isomeric modifications such as pseudouridine (Ψ) from uridine (U). Our recent study incorporates a two-dimensional hydrophobic end labeling strategy into MS-based sequencing (2D-HELS MS Seq) to systematically address the current challenges in MS sequencing of RNA, making it possible to directly and de novo sequence purified single RNA and mixed RNA containing both canonical and modified nucleotides. Here, we describe the method to sequence representative single-RNA and mixed-RNA oligonucleotides, each with a different sequence and/or containing modified nucleotides such as Ψ and 5-methylcytosine (m5C), using 2D-HELS MS Seq.
Assuntos
Cromatografia Líquida/métodos , Nucleotídeos/genética , RNA/genética , Análise de Sequência de RNA/métodos , Espectrometria de Massas em Tandem/métodos , 5-Metilcitosina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos/genética , Pseudouridina/genética , Processamento Pós-Transcricional do RNA/genética , Uridina/genéticaRESUMO
SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.
RESUMO
In our previous study, three kinds of grapefruit peel soluble dietary fibers (SDFs) were prepared by microwave-assisted modifications, including microwave-sodium hydroxide treatment SDF (MST-SDF), microwave-enzymatic treatment SDF (MET-SDF) and microwave-ultrasonic treatment SDF (MUT-SDF). The present study aimed to investigate the structural, functional and in vitro digestion properties of three kinds of bread incorporated with SDFs, named MST-SDF bread (SB), MET-SDF bread (EB), and MUT-SDF bread (UB). Scanning electron microscopy, X-ray diffraction, differential scanning calorimetry and texture profile analysis were used to determine the structural properties. In comparison with the SB and EB, the UB showed an ideal hardness and internal structure, and also significant improvement of the thermal stability. Meanwhile, the UB exhibited the highest overall acceptability in the sensory evaluation. In addition, the water holding capacity (WHC), cholesterol adsorption capacity (CAC) and nitrite ion adsorption capacity (NIAC) of UB were superior to those of the other two samples. Moreover, the in vitro digestive glucose release rates of breads were all significantly reduced by the addition of SDFs, especially that of UB. In summary, the present study showed that UB presented the best performance in terms of structural, functional and in vitro digestion properties, implying that MUT-SDF could be utilized in bread with high quality and low glucose release rate, and developed as a potential ingredient of functional food.
Assuntos
Pão/análise , Citrus paradisi/química , Fibras na Dieta/análise , Digestão , Adsorção , Varredura Diferencial de Calorimetria , China , Colesterol/análise , Feminino , Dureza , Humanos , Masculino , Microscopia Eletrônica de Varredura , Micro-Ondas , Hidróxido de Sódio , Paladar , Ultrassom , Água/química , Difração de Raios XRESUMO
Post-transcriptional modifications are intrinsic to RNA structure and function. However, methods to sequence RNA typically require a cDNA intermediate and are either not able to sequence these modifications or are tailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with <100% frequency at their particular sites, and site-specific quantification of their stoichiometries is another challenge. Here, we report a direct method for sequencing tRNAPhe without cDNA by integrating a two-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNAPhe was sequenced and the identity, location, and stoichiometry of all eleven different RNA modifications was determined, five of which were not 100% modified, including a 2'-O-methylated G (Gm) in the wobble anticodon position as well as an N2, N2-dimethylguanosine (m22G), a 7-methylguanosine (m7G), a 1-methyladenosine (m1A), and a wybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3'-CCA tail of the tRNAPhe (75 nt with a 3'-CC tail (80% abundance) and 74 nt with a 3'-C tail (3% abundance)) were identified in addition to the full-length 3'-CCA-tailed tRNAPhe (76 nt, 17% abundance). We discovered a new isoform with A-G transitions/editing at the 44 and 45 positions in the tRNAPhe variable loop, and discuss possible mechanisms related to the emergence and functions of the isoforms with these base transitions or editing. Our method revealed new isoforms, base modifications, and RNA editing as well as their stoichiometries in the tRNA that cannot be determined by current cDNA-based methods, opening new opportunities in the field of epitranscriptomics.