RESUMEN
Viral diseases pose a serious global health threat due to their rapid transmission and widespread impact. The RNA-dependent RNA polymerase (RdRp) participates in the synthesis, transcription, and replication of viral RNA in host. The current study investigates the antiviral potential of secondary metabolites particularly those derived from bacteria, fungi, and plants to develop novel medicines. Using a virtual screening approach that combines molecular docking and molecular dynamics (MD) simulations, we aimed to discover compounds with strong interactions with RdRp of five different retroviruses. The top five compounds were selected for each viral RdRp based on their docking scores, binding patterns, molecular interactions, and drug-likeness properties. The molecular docking study uncovered several metabolites with antiviral activity against RdRp. For instance, cytochalasin Z8 had the lowest docking score of -8.9 (kcal/mol) against RdRp of SARS-CoV-2, aspulvinone D (-9.2 kcal/mol) against HIV-1, talaromyolide D (-9.9 kcal/mol) for hepatitis C, aspulvinone D (-9.9 kcal/mol) against Ebola and talaromyolide D also maintained the lowest docking score of -9.2 kcal/mol against RdRp enzyme of dengue virus. These compounds showed remarkable antiviral potential comparable to standard drug (remdesivir -7.4 kcal/mol) approved to target RdRp and possess no significant toxicity. The molecular dynamics simulation confirmed that the best selected ligands were firmly bound to their respective target proteins for a simulation time of 200 ns. The identified lead compounds possess distinctive pharmacological characteristics, making them potential candidates for repurposing as antiviral drugs against SARS-CoV-2. Further experimental evaluation and investigation are recommended to ascertain their efficacy and potential.
Asunto(s)
Antivirales , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , ARN Polimerasa Dependiente del ARN , Antivirales/farmacología , Antivirales/química , Antivirales/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Humanos , VIH-1/efectos de los fármacos , VIH-1/enzimología , Metabolismo Secundario , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/metabolismoRESUMEN
Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO2-TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO2-TSC. Moreover, resistance selection and characterization showed that N4-TSCR mutants were highly resistant to N4-TSC but remained susceptible to 6NO2-TSC. In contrast, 6NO2-TSCR mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSCR mutants, whereas I261 M was found in 6NO2-TSCR mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a "hot spot" for BVDV non-nucleoside inhibitors.
Asunto(s)
Antivirales , Farmacorresistencia Viral , Genotipo , Indanos , Tiosemicarbazonas , Antivirales/farmacología , Antivirales/química , Animales , Bovinos , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/química , Indanos/farmacología , Indanos/química , Farmacorresistencia Viral/genética , Virus de la Diarrea Viral Bovina Tipo 1/efectos de los fármacos , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Virus de la Diarrea Viral Bovina/genética , Línea Celular , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Virus de la Diarrea Viral Bovina Tipo 2/genética , Virus de la Diarrea Viral Bovina Tipo 2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Mutación , ARN Viral/genéticaRESUMEN
The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA-dependent RNA polymerase (RdRp) has emerged as a potential target for broad-spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently-used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring-opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad-spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high-throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 µM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 µM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 µM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 µM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring-opening" bases and suggests the "ring-opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open-loop bases to mimic Watson-Crick base pairing better and improve antiviral activity.
Asunto(s)
Antivirales , Diseño de Fármacos , Nucleósidos , ARN Polimerasa Dependiente del ARN , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Nucleósidos/química , Nucleósidos/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Humanos , Animales , Células de Riñón Canino Madin Darby , Perros , Relación Estructura-ActividadRESUMEN
Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is an important pathogen affecting cloven-hoof livestock. However, neither effective vaccines covering all serotypes nor specific antivirals against FMDV infections are currently available. In this study, we employed virtual screening to screen for secondary metabolite terpenoids targeting the RNA-dependent RNA polymerase (RdRp), or 3Dpol, of FMDV. Subsequently, we identified the potential antiviral activity of the 32 top-ranked terpenoids, revealing that continentalic acid, dehydroabietic acid (abietic diterpenoids), brusatol, bruceine D, and bruceine E (tetracyclic triterpenoids) significantly reduced cytopathic effects and viral infection in the terpenoid-treated, FMDV-infected BHK-21 cells in a dose-dependent manner, with nanomolar to low micromolar levels. The FMDV minigenome assay demonstrated that brusatol and bruceine D, in particular, effectively blocked FMDV 3Dpol activity, exhibiting IC50 values in the range of 0.37-0.39 µM and surpassing the efficacy of the antiviral drug control, ribavirin. Continentalic acid and bruceine E exhibited moderate inhibition of FMDV 3Dpol. The predicted protein-ligand interaction confirmed that these potential terpenoids interacted with the main catalytic and bystander residues of FMDV 3Dpol. Additionally, brusatol and bruceine D exhibited additive effects when combined with ribavirin. In conclusion, terpenoids from natural resources show promise for the development of anti-FMD agents.
Asunto(s)
Antivirales , Virus de la Fiebre Aftosa , Terpenos , Virus de la Fiebre Aftosa/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Animales , Terpenos/farmacología , Terpenos/química , Línea Celular , Replicación Viral/efectos de los fármacos , Simulación por Computador , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Cricetinae , Simulación del Acoplamiento Molecular , Fiebre Aftosa/virología , Fiebre Aftosa/tratamiento farmacológico , Diterpenos/farmacología , Diterpenos/químicaRESUMEN
COVID-19 caused major worldwide problems. The spread of variants and limited treatment encouraged the design of novel anti-SARS-CoV-2 compounds. A series of compounds RH1-23 were designed to dually target RNA-dependent RNA polymerase (RdRp) and transmembrane serine protease 2 (TMPRSS2). Compared to remdesivir, in vitro screening indicated the highest selectivity and potent activity of RH11-13 with half maximum inhibitory concentration (IC50) 3.9, 5.7, and 19.72 nM, respectively. RH11-12 showed superior inhibition activity against TMPRSS2 and RdRP with IC50 (1.7 and 4.2), and (6.1 and 4.42) nM, respectively. WaterMap analysis and molecular dynamics studies demonstrated the superior enzyme binding activity of RH11 and RH12. On Vero-E6 cells, RH11 and RH12 significantly inhibited the viral replication with 66 % and 63.2 %, and viral adsorption with 44 % and 65 %, alongside virucidal effect with 51.40 % and 90.5 %, respectively. Furthermore, the potent activity of RH12 was tested on TMPRSS2-expressing cells (Calu-3) compared to camostat. RH12 exhibited selectivity index (26.05) similar to camostat (28.01) and comparable to its SI on Vero-E6 cells (22.6). RH12 demonstrated also a significant inhibition of the viral adsorption on Calu-3 cells with 60 % inhibition at 30 nM. The designed compounds exhibited good physiochemical properties. These findings indicate a broad-spectrum antiviral efficacy of the designed compounds, particularly RH12, with a promise for further development.
Asunto(s)
Antivirales , SARS-CoV-2 , Serina Endopeptidasas , Serina Endopeptidasas/metabolismo , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Chlorocebus aethiops , Animales , Células Vero , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19 , Relación Estructura-Actividad , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Dosis-Respuesta a Droga , Estructura Molecular , Simulación de Dinámica MolecularRESUMEN
The spread of the influenza virus has caused devastating pandemics and huge economic losses worldwide. Antiviral drugs with diverse action modes are urgently required to overcome the challenges of viral mutation and drug resistance, and targeted protein degradation strategies constitute excellent candidates for this purpose. Herein, the first degradation of the influenza virus polymerase acidic (PA) protein using small-molecule degraders developed by hydrophobic tagging (HyT) technology to effectively combat the influenza virus was reported. The SAR results revealed that compound 19b with Boc2-(L)-Lys demonstrated excellent inhibitory activity against A/WSN/33/H1N1 (EC50 = 0.015 µM) and amantadine-resistant strain (A/PR/8/H1N1), low cytotoxicity, high selectivity, substantial degradation ability, and good drug-like properties. Mechanistic studies demonstrated that the proteasome system and autophagic lysosome pathway were the potential drivers of these HyT degraders. Thus, this study provides a powerful tool for investigating the targeted degradation of influenza virus proteins and for antiviral drug development.
Asunto(s)
Antivirales , Interacciones Hidrofóbicas e Hidrofílicas , Tiourea , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Humanos , Perros , Animales , Tiourea/farmacología , Tiourea/análogos & derivados , Tiourea/química , Relación Estructura-Actividad , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Células de Riñón Canino Madin Darby , Proteolisis/efectos de los fármacos , Proteínas Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Farmacorresistencia Viral/efectos de los fármacosRESUMEN
RNA-dependent RNA polymerase (RdRp) is considered a potential drug target for dengue virus (DENV) inhibition and has attracted attention in antiviral drug discovery. Here, we screened 121 natural compounds from Litsea cubeba against DENV RdRp using various approaches of computer-based drug discovery. Notably, we identified four potential compounds (Ushinsunine, Cassameridine, (+)-Epiexcelsin, (-)-Phanostenine) with good binding scores and allosteric interactions with the target protein. Moreover, molecular dynamics simulation studies were done to check the conformational stability of the complexes under given conditions. Additionally, we performed post-simulation analysis to find the stability of potential drugs in the target protein. The findings suggest Litsea cubeba-derived phytomolecules as a therapeutic solution to control DENV infection.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Antivirales , Virus del Dengue , Litsea , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fitoquímicos , ARN Polimerasa Dependiente del ARN , Virus del Dengue/efectos de los fármacos , Virus del Dengue/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Antivirales/farmacología , Antivirales/química , Fitoquímicos/farmacología , Fitoquímicos/química , Regulación Alostérica/efectos de los fármacos , Litsea/química , Unión ProteicaRESUMEN
Background: The RNA-dependent RNA polymerase (RdRp) inhibitors, molnupiravir and VV116, have the potential to maximize clinical benefits in the oral treatment of COVID-19. Subjects who consume these drugs may experience an increased incidence of adverse events. This study aimed to evaluate the safety profile of molnupiravir and VV116. Methods: A comprehensive search of scientific and medical databases, such as PubMed Central/Medline, Embase, Web of Science, and Cochrane Library, was conducted to find relevant articles in English from January 2020 to June 2023. Any kind of adverse events reported in the study were pooled and analyzed in the drug group versus the control group. Estimates of risk effects were summarized through the random effects model using Review Manager version 5.2, and sensitivity analysis was performed by Stata 17.0 software. Results: Fifteen studies involving 32,796 subjects were included. Eleven studies were placebo-controlled, and four were Paxlovid-controlled. Twelve studies reported adverse events for molnupiravir, and three studies described adverse events for VV116. The total odds ratio (OR) for adverse events in the RdRp inhibitor versus the placebo-controlled group was 1.01 (95% CI=0.84-1.22; I2=26%), P=0.88. The total OR for adverse events in the RdRp inhibitor versus the Paxlovid-controlled group was 0.32 (95% CI=0.16-0.65; I2=87%), P=0.002. Individual drug subgroup analysis in the placebo-controlled study showed that compared with the placebo group, a total OR for adverse events was 0.97 (95% CI, 0.85-1.10; I2=0%) in the molnupiravir group and 3.77 (95% CI=0.08-175.77; I2=85%) in the VV116 group. Conclusion: The RdRp inhibitors molnupiravir and VV116 are safe for oral treatment of COVID-19. Further evidence is necessary that RdRp inhibitors have a higher safety profile than Paxlovid.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , Citidina , Hidroxilaminas , ARN Polimerasa Dependiente del ARN , Humanos , Hidroxilaminas/uso terapéutico , Hidroxilaminas/farmacología , Citidina/análogos & derivados , Citidina/uso terapéutico , Citidina/farmacología , Antivirales/uso terapéutico , Antivirales/efectos adversos , Antivirales/farmacología , Administración Oral , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2 , Adenosina/análogos & derivadosRESUMEN
Dialkyl/aryl aminophosphonates, 3a-g and 4a-e were synthesized using the LiClO4 catalyzed Kabachnic Fields-type reaction straightforwardly and efficiently. The synthesized phosphonates structures were characterized using elemental analyses, FT-IR, 1H NMR, 13C NMR, and MS spectroscopy. The new compounds were subjected to in-silico molecular docking simulations to evaluate their potential inhibition against Influenza A Neuraminidase and RNA-dependent RNA polymerase of human coronavirus 229E. Subsequently, the compounds were further tested in vitro using a cytopathic inhibition assay to assess their antiviral activity against both human Influenza (H1N1) and human coronavirus (HCoV-229E). Diphenyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (furan-2-yl) methyl) phosphonate (3f) and diethyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) methyl) phosphonate (4e) were demonstrated direct inhibition activity against Influenza A Neuraminidase and RNA-dependent RNA polymerase. This was supported by their highly favorable binding energies in-silico, with top-ranked values of -12.5 kcal/mol and -14.2 kcal/mol for compound (3f), and -13.5 kcal/mol and -9.89 kcal/mol for compound (4e). Moreover, they also displayed notable antiviral efficacy in vitro against both viruses. These compounds demonstrated significant antiviral activity, as evidenced by selectivity indices (SI) of 101.7 and 51.8, respectively against H1N1, and 24.5 and 5.1 against HCoV-229E, respectively.
Asunto(s)
Antivirales , Coronavirus Humano 229E , Diseño de Fármacos , Subtipo H1N1 del Virus de la Influenza A , Simulación del Acoplamiento Molecular , Organofosfonatos , Pirimidinonas , Antivirales/farmacología , Antivirales/síntesis química , Antivirales/química , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Humanos , Pirimidinonas/farmacología , Pirimidinonas/síntesis química , Pirimidinonas/química , Relación Estructura-Actividad , Organofosfonatos/farmacología , Organofosfonatos/química , Organofosfonatos/síntesis química , Coronavirus Humano 229E/efectos de los fármacos , Estructura Molecular , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/metabolismo , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismoRESUMEN
Human norovirus (HuNoV), a primary cause of non-bacterial gastroenteritis, currently lacks approved treatment. RdRp is vital for virus replication, making it an attractive target for therapeutic intervention. By application of structure-based virtual screening procedure, we present CX-6258 hydrochloride hydrate as a potent RdRp non-nucleoside inhibitor, effectively inhibiting HuNoV RdRp activity with an IC50 of 3.61 µM. Importantly, this compound inhibits viral replication in cell culture, with an EC50 of 0.88 µM. In vitro binding assay validate that CX-6258 hydrochloride hydrate binds to RdRp through interaction with the "B-site" binding pocket. Interestingly, CX-6258-contacting residues such as R392, Q439, and Q414 are highly conserved among major norovirus GI and GII variants, suggesting that it may be a general inhibitor of norovirus RdRp. Given that CX-6258 hydrochloride hydrate is already utilized as an orally efficacious pan-Pim kinase inhibitor, it may serve as a potential lead compound in the effort to control HuNoV infections.
Asunto(s)
Antivirales , Norovirus , ARN Polimerasa Dependiente del ARN , Replicación Viral , Norovirus/efectos de los fármacos , Norovirus/enzimología , Norovirus/genética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Antivirales/farmacología , Antivirales/química , Humanos , Replicación Viral/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Sitios de UniónRESUMEN
Long antisense RNAs (asRNAs) have been observed to repress HIV and other virus expression in a manner that is refractory to viral evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) disease, has a distinct ability to evolve resistance around antibody targeting, as was evident from the emergence of various SARS-CoV-2 spike antibody variants. Importantly, the effectiveness of current antivirals is waning due to the rapid emergence of new variants of concern, more recently the omicron variant. One means of avoiding the emergence of viral resistance is by using long asRNA to target SARS-CoV-2. Similar work has proven successful with HIV targeting by long asRNA. In this study, we describe a long asRNA targeting SARS-CoV-2 RNA-dependent RNA polymerase gene and the ability to deliver this RNA in extracellular vesicles (EVs) to repress virus expression. The observations presented in this study suggest that EV-delivered asRNAs are one means to targeting SARS-CoV-2 infection, which is both effective and broadly applicable as a means to control viral expression in the absence of mutation. This is the first demonstration of the use of engineered EVs to deliver long asRNA payloads for antiviral therapy.
Asunto(s)
COVID-19 , Vesículas Extracelulares , ARN sin Sentido , SARS-CoV-2 , Vesículas Extracelulares/genética , Vesículas Extracelulares/virología , Vesículas Extracelulares/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/efectos de los fármacos , Humanos , ARN sin Sentido/genética , ARN sin Sentido/uso terapéutico , COVID-19/virología , COVID-19/genética , COVID-19/terapia , Animales , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Células Vero , Chlorocebus aethiops , Antivirales/uso terapéutico , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19RESUMEN
COVID-19, a disease caused by SARS-CoV-2, has caused a huge loss of human life, and the number of deaths is still continuing. Despite the lack of repurposed drugs and vaccines, the search for potential small molecules to inhibit SARS-CoV-2 is in demand. Hence, we relied on the drug-like characters of ten phytochemicals (compounds 1-10) that were previously isolated and purified by our research team from Saudi medicinal plants. We computationally evaluated the inhibition of RNA-dependent RNA polymerase (RdRp) by compounds 1-10. Non-covalent (reversible) docking of compounds 1-10 with RdRp led to the formation of a hydrogen bond with template primer nucleotides (A and U) and key amino acid residues (ASP623, LYS545, ARG555, ASN691, SER682, and ARG553) in its active pocket. Covalent (irreversible) docking revealed that compounds 7, 8, and 9 exhibited their irreversible nature of binding with CYS813, a crucial amino acid in the palm domain of RdRP. Molecular dynamic (MD) simulation analysis by RMSD, RMSF, and Rg parameters affirmed that RdRP complexes with compounds 7, 8, and 9 were stable and showed less deviation. Our data provide novel information on compounds 7, 8, and 9 that demonstrated their non-nucleoside and irreversible interaction capabilities to inhibit RdRp and shed new scaffolds as antivirals against SARS-CoV-2.
Asunto(s)
Antivirales , Plantas Medicinales , ARN Polimerasa Dependiente del ARN , SARS-CoV-2 , Aminoácidos , Antivirales/farmacología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plantas Medicinales/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Arabia SauditaRESUMEN
Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and encephalitis in humans and animals, and are considered a substantial health and economic burden worldwide1. Replication and transcription of the viral genome are executed by the large (L) polymerase, which is a promising target for the development of antiviral drugs. Here, using the L polymerase of EBOV as a representative, we show that de novo replication of L polymerase is controlled by the specific 3' leader sequence of the EBOV genome in an enzymatic assay, and that formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence. We present the high-resolution structures of the EBOV L-VP35-RNA complex and show that the 3' leader RNA binds in the template entry channel with a distinctive stable bend conformation. Using mutagenesis assays, we confirm that the bend conformation of the RNA is required for the de novo replication activity and reveal the key residues of the L protein that stabilize the RNA conformation. These findings provide a new mechanistic understanding of RNA synthesis for polymerases of non-segmented negative-strand RNA viruses, and reveal important targets for the development of antiviral drugs.
Asunto(s)
Ebolavirus , ARN Viral , ARN Polimerasa Dependiente del ARN , Replicación Viral , Animales , Humanos , Antivirales/farmacología , Ebolavirus/enzimología , Ebolavirus/genética , Ebolavirus/crecimiento & desarrollo , Fiebre Hemorrágica Ebola/virología , ARN Viral/biosíntesis , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Genoma Viral , Conformación de Ácido Nucleico , Mutagénesis , Estabilidad del ARNRESUMEN
A series of thiazole linked Oxindole-5-Sulfonamide (OSA) derivatives were designed as inhibitors of RNA-dependent RNA polymerase (RdRp) activity of Dengue virus. These were synthesized and then evaluated for their efficacy in ex-vivo virus replication assay using human cell lines. Among 20 primary compounds in the series, OSA-15 was identified as a hit. A series of analogues were synthesized by replacing the difluoro benzyl group of OSA-15 with different substituted benzyl groups. The efficacy of OSA-15derivatives was less than that of the parent compound, except OSA-15-17, which has shown improved efficacy than OSA-15. The further optimization was carried out by adding dimethyl (DM) groups to both the sulfonamide and oxindole NH's to produce OSA-15-DM and OSA-15-17-DM. These two compounds were showing no detectable cytotoxicity and the latter was more efficacious. Further, both these compounds were tested for inhibition in all the serotypes of the Dengue virus using an ex-vivo assay. The EC50 of OSA-15-17-DM was observed in a low micromolar range between 2.5 and 5.0 µg/ml. Computation docking and molecular dynamics simulation studies confirmed the binding of identified hits to DENV RdRp. OSA15-17-DM blocks the RNA entrance and elongation site for their biological activity with high binding affinity. Overall, the identified oxindole derivatives are novel compounds that can inhibit Dengue replication, working as non-nucleoside inhibitors (NNI) to explore as anti-viral RdRp activity.
Asunto(s)
Antivirales , Dengue , Oxindoles , Antivirales/química , Dengue/tratamiento farmacológico , Virus del Dengue , Simulación del Acoplamiento Molecular , Oxindoles/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sulfonamidas/farmacologíaRESUMEN
Numerous pathogens affecting human is present in the flavivirus family namely west nile, dengue, yellow fever, and zika which involves in development of global burden and distressing the environment economically. Till date, no approved drugs are available for targeting these viruses. The threat which urged the identification of small molecules for the inhibition of these viruses is the spreading of serious viral diseases. The recent outbreak of zika and dengue infections postured a solemn risk to worldwide public well-being. RNA-dependent RNA polymerase (RdRp) is the supreme adaptable enzymes of all the RNA viruses which is responsible for the replication and transcription of genome among the structural and nonstructural proteins of flaviviruses. It is understood that the RdRp of the flaviviruses are similar stating that the japanese encephalitis and west nile shares 70% identity with zika whereas the dengue serotype 2 and 3 shares the identity of 76% and 81%, respectively. In this study, we investigated the binding site of four flaviviral RdRp and provided insights into various interaction of the molecules using the computational approach. Our study helps in recognizing the potent compounds that could inhibit the viral protein as a common inhibitor. Additionally, with the conformational stability analysis, we proposed the possible mechanism of inhibition of the identified common small molecule toward RdRp of flavivirus. Finally, this study could be an initiative for the identification of common inhibitors and can be explored further for understanding the mechanism of action through in vitro studies for the study on efficacy.
Asunto(s)
Reposicionamiento de Medicamentos , Flavivirus , ARN Polimerasa Dependiente del ARN , Humanos , Dengue/tratamiento farmacológico , Flavivirus/efectos de los fármacos , Flavivirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Virus Zika/efectos de los fármacos , Virus Zika/enzimología , Infección por el Virus Zika/tratamiento farmacológicoRESUMEN
Study Background & Objective: After the influenza pandemic (1918), COVID-19 was declared a Vth pandemic by the WHO in 2020. SARS-CoV-2 is an RNA-enveloped single-stranded virus. Based on the structure and life cycle, Protease (3CLpro), RdRp, ACE2, IL-6, and TMPRSS2 are the major targets for drug development against COVID-19. Pre-existing several drugs (FDA-approved) are used to inhibit the above targets in different diseases. In coronavirus treatment, these drugs are also in different clinical trial stages. Remdesivir (RdRp inhibitor) is the only FDA-approved medicine for coronavirus treatment. In the present study, by using the drug repurposing strategy, 70 preexisting clinical or under clinical trial molecules were used in scrutiny for RdRp inhibitor potent molecules in coronavirus treatment being surveyed via docking studies. Molecular simulation studies further confirmed the binding mechanism and stability of the most potent compounds. MATERIAL AND METHODS: Docking studies were performed using the Maestro 12.9 module of Schrodinger software over 70 molecules with RdRp as the target and remdesivir as the standard drug and further confirmed by simulation studies. RESULTS: The docking studies showed that many HIV protease inhibitors demonstrated remarkable binding interactions with the target RdRp. Protease inhibitors such as lopinavir and ritonavir are effective. Along with these, AT-527, ledipasvir, bicalutamide, and cobicistat showed improved docking scores. RMSD and RMSF were further analyzed for potent ledipasvir and ritonavir by simulation studies and were identified as potential candidates for corona disease. CONCLUSION: The drug repurposing approach provides a new avenue in COVID-19 treatment.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19 , ARN Polimerasa Dependiente del ARN , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Antivirales/química , Tratamiento Farmacológico de COVID-19/métodos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Ritonavir , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/metabolismoRESUMEN
The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3'-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.
Asunto(s)
COVID-19 , Citidina Trifosfato , ARN Polimerasa Dependiente del ARN , SARS-CoV-2 , Humanos , Antivirales/farmacología , Antivirales/química , Microscopía por Crioelectrón , Citidina Trifosfato/química , Simulación de Dinámica Molecular , Nucleósidos , Nucleótidos , Ribosa , ARN Viral , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/química , SARS-CoV-2/metabolismoRESUMEN
The World Health Organization has declared the novel coronavirus (COVID-19) outbreak a global pandemic and emerging threat to people in the 21st century. SARS-CoV-2 constitutes RNA-Dependent RNA Polymerase (RdRp) viral proteins, a critical target in the viral replication process. No FDA-approved drug is currently available, and there is a high demand for therapeutic strategies against COVID-19. In search of the anti-COVID-19 compound from traditional medicine, we evaluated the active moieties from Nilavembu Kudineer (NK), a poly-herbal Siddha formulation recommended by AYUSH against COVID-19. We conducted a preliminary docking analysis of 355 phytochemicals (retrieved from PubChem and IMPPAT databases) present in NK against RdRp viral protein (PDB ID: 7B3B) using COVID-19 Docking Server and further with AutoDockTool-1.5.6. MD simulation studies confirmed that Orientin (L1), Vitexin (L2), and Kasuagamycin (L3) revealed better binding activity against RdRp (PDB ID: 7B3B) in comparison with Remdesivir. The study suggests a potential scaffold for developing drug candidates against COVID-19. PRACTICAL APPLICATIONS: Nilavembu Kudineer is a poly-herbal Siddha formulation effective against various diseases like cough, fever, breathing problems, etc. This study shows that different phytoconstituents identified from Nilavembu Kudineer were subjected to in silico and ADME analyses. Out of the former 355 phytochemical molecules, Orientin (L1), Vitexin (L2), and Kasuagamycin (L3) showed better binding activity against RdRp viral protein (PDB ID: 7B3B) in comparison with the synthetic repurposed drug. Our work explores the search for an anti-COVID-19 compound from traditional medicine like Nilavembu Kudineer, which can be a potential scaffold for developing drug candidates against COVID-19.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Preparaciones de Plantas , ARN Polimerasa Dependiente del ARN , Humanos , Simulación del Acoplamiento Molecular , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Proteínas Virales , Replicación Viral/efectos de los fármacos , Medicina Ayurvédica , Preparaciones de Plantas/uso terapéuticoRESUMEN
Nucleotide/nucleoside analogs (NAs) are important compounds used in antiviral drug development. To understand the action mode of NA drugs, we present an enzymology protocol to initially evaluate the intervention mechanism of the NTP forms of NAs on a coronaviral RNA-dependent RNA polymerase (RdRP). We describe the preparation of SARS-CoV-2 RdRP proteins and RNA constructs, followed by a primer-dependent RdRP assay to assess NTP forms of NAs. Two representative NA drugs, sofosbuvir and remdesivir, are used for demonstration of this protocol. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).
Asunto(s)
Nucleósidos , Nucleótidos , ARN Polimerasa Dependiente del ARN , SARS-CoV-2 , Nucleósidos/análogos & derivados , Nucleósidos/farmacología , Nucleótidos/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recently emerged human coronavirus. COVID-19 vaccines have proven to be successful in protecting the vaccinated from infection, reducing the severity of disease, and deterring the transmission of infection. However, COVID-19 vaccination faces many challenges, such as the decline in vaccine-induced immunity over time, and the decrease in potency against some SARS-CoV-2 variants including the recently emerged Omicron variant, resulting in breakthrough infections. The challenges that COVID-19 vaccination is facing highlight the importance of the discovery of antivirals to serve as another means to tackle the pandemic. To date, neutralizing antibodies that block viral entry by targeting the viral spike protein make up the largest class of antivirals that has received US FDA emergency use authorization (EUA) for COVID-19 treatment. In addition to the spike protein, other key targets for the discovery of direct-acting antivirals include viral enzymes that are essential for SARS-CoV-2 replication, such as RNA-dependent RNA polymerase and proteases, as judged by US FDA approval for remdesivir, and EUA for Paxlovid (nirmatrelvir + ritonavir) for treating COVID-19 infections. This review presents an overview of the current status and future direction of antiviral drug discovery for treating SARS-CoV-2 infections, covering important antiviral targets such as the viral spike protein, non-structural protein (nsp) 3 papain-like protease, nsp5 main protease, and the nsp12/nsp7/nsp8 RNA-dependent RNA polymerase complex.