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1.
Enzymes ; 49: 315-354, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696837

RESUMO

The treatment of viral infections remains challenging, in particular in the face of emerging pathogens. Broad-spectrum antiviral drugs could potentially be used as a first line of defense. The RNA-dependent RNA polymerase (RdRp) of RNA viruses serves as a logical target for drug discovery and development efforts. Herein we discuss compounds that target RdRp of poliovirus, hepatitis C virus, influenza viruses, respiratory syncytial virus, and the growing data on coronaviruses. We focus on nucleotide analogs and mechanisms of action and resistance.


Assuntos
Antivirais/farmacologia , Nucleotídeos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas do Complexo da Replicase Viral/antagonistas & inibidores , Vírus/enzimologia , Replicação Viral
2.
Int J Mol Sci ; 22(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34681802

RESUMO

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new pathogen from the family of Coronaviridae that caused a global pandemic of COVID-19 disease. In the absence of effective antiviral drugs, research of novel therapeutic targets such as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) becomes essential. This viral protein is without a human counterpart and thus represents a unique prospective drug target. However, in vitro biological evaluation testing on RdRp remains difficult and is not widely available. Therefore, we prepared a database of commercial small-molecule compounds and performed an in silico high-throughput virtual screening on the active site of the SARS-CoV-2 RdRp using ensemble docking. We identified a novel thioether-amide or guanidine-linker class of potential RdRp inhibitors and calculated favorable binding free energies of representative hits by molecular dynamics simulations coupled with Linear Interaction Energy calculations. This innovative procedure maximized the respective phase-space sampling and yielded non-covalent inhibitors representing small optimizable molecules that are synthetically readily accessible, commercially available as well as suitable for further biological evaluation and mode of action studies.


Assuntos
Antivirais/química , Inibidores Enzimáticos/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/enzimologia , Proteínas Virais/antagonistas & inibidores , Amidas/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/virologia , Domínio Catalítico , Bases de Dados de Compostos Químicos , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Guanidina/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/isolamento & purificação , Relação Estrutura-Atividade , Sulfetos/química , Termodinâmica , Proteínas Virais/metabolismo
3.
Nat Commun ; 12(1): 5233, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475387

RESUMO

Measles virus (MeV) is a highly contagious pathogen that enters the human host via the respiratory route. Besides acute pathologies including fever, cough and the characteristic measles rash, the infection of lymphocytes leads to substantial immunosuppression that can exacerbate the outcome of infections with additional pathogens. Despite the availability of effective vaccine prophylaxis, measles outbreaks continue to occur worldwide. We demonstrate that prophylactic and post-exposure therapeutic treatment with an orally bioavailable small-molecule polymerase inhibitor, ERDRP-0519, prevents measles disease in squirrel monkeys (Saimiri sciureus). Treatment initiation at the onset of clinical signs reduced virus shedding, which may support outbreak control. Results show that this clinical candidate has the potential to alleviate clinical measles and augment measles virus eradication.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Sarampo/prevenção & controle , Morfolinas/uso terapêutico , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacocinética , Tolerância Imunológica/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Vírus do Sarampo/efeitos dos fármacos , Morfolinas/farmacocinética , Piperidinas/farmacocinética , Pirazóis/farmacocinética , Saimiri , Replicação Viral/efeitos dos fármacos , Eliminação de Partículas Virais/efeitos dos fármacos
4.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
5.
Int Immunopharmacol ; 98: 107831, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34247016

RESUMO

Explicit hindrance and blockade of the viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is considered one of the most promising and efficient approaches for developing highly potent remedies for COVID-19. However, almost all of the reported viral RdRp inhibitors (either repurposed or new antiviral drugs) lack specific selectivity against the novel coronaviral RdRp and still at a beginning phase of advancement. Herein, I discovered and introduce a new pyrazine derivative, (E)-N-(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide (cyanorona-20), as the first potent SARS-CoV-2 RdRp inhibitor with very high selectivity (209- and 45-fold more potent than favipiravir and remdesivir, respectively). This promising selective specific anti-COVID-19 compound is also deemed to be the first distinctive derivative of favipiravir. Cyanorona-20, the unprecedented nucleoside/nucleotide analog, was designed, synthesized, characterized, computationally studied, and biologically evaluated for its anti-COVID-19 actions (through a precise in vitro anti-COVID-19 assay). The results of the biological assay displayed that cyanorona-20 surprisingly exhibited very high and largely significant anti-COVID-19 activities (anti-SARS-CoV-2 EC50 = 0.45 µM), and, in addition, it could be also a very promising guide and lead compound for the design and synthesis of new anti-SARS-CoV-2 and anti-COVID-19 agents through structural modifications and further computational studies. Further appraisal for the improvement of cyanorona-20 medication is a prerequisite requirement in the coming days. In a word, the ascent of the second member (cyanorona-20 "Corona Antidote") of the novel and promising class of anti-COVID-19 pyrazine derivatives would drastically make a medical uprising in the pharmacotherapeutic treatment regimens and protocols of the recently-emerged SARS-CoV-2 infection and its accompanying COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , COVID-19/diagnóstico , COVID-19/virologia , Desenho Assistido por Computador , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Terapia de Alvo Molecular , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/crescimento & desenvolvimento , Relação Estrutura-Atividade
6.
PLoS One ; 16(7): e0253364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270554

RESUMO

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.


Assuntos
Antivirais/farmacologia , Inibidores de Proteases/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Anticorpos de Cadeia Única/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Células A549 , Complexo Antígeno-Anticorpo , Humanos , Concentração Inibidora 50 , RNA Polimerase Dependente de RNA/imunologia , RNA Polimerase Dependente de RNA/metabolismo , Anticorpos de Cadeia Única/imunologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo
7.
Molecules ; 26(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205768

RESUMO

Since December 2019, novel coronavirus disease 2019 (COVID-19) pandemic has caused tremendous economic loss and serious health problems worldwide. In this study, we investigated 14 natural compounds isolated from Amphimedon sp. via a molecular docking study, to examine their ability to act as anti-COVID-19 agents. Moreover, the pharmacokinetic properties of the most promising compounds were studied. The docking study showed that virtually screened compounds were effective against the new coronavirus via dual inhibition of SARS-CoV-2 RdRp and the 3CL main protease. In particular, nakinadine B (1), 20-hepacosenoic acid (11) and amphimedoside C (12) were the most promising compounds, as they demonstrated good interactions with the pockets of both enzymes. Based on the analysis of the molecular docking results, compounds 1 and 12 were selected for molecular dynamics simulation studies. Our results showed Amphimedon sp. to be a rich source for anti-COVID-19 metabolites.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Proteases 3C de Coronavírus/química , Poríferos/química , Poríferos/metabolismo , RNA Polimerase Dependente de RNA/química , SARS-CoV-2/efeitos dos fármacos , Amino Açúcares/química , Amino Açúcares/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacocinética , COVID-19/tratamento farmacológico , Biologia Computacional , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Piridinas/química , Piridinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/metabolismo
8.
Biomed Pharmacother ; 138: 111544, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34311539

RESUMO

The RNA-dependent RNA polymerase (RdRp) and 3C-like protease (3CLpro) from SARS-CoV-2 play crucial roles in the viral life cycle and are considered the most promising targets for drug discovery against SARS-CoV-2. In this study, FDA-approved drugs were screened to identify the probable anti-RdRp and 3CLpro inhibitors by molecular docking approach. The number of ligands selected from the PubChem database of NCBI for screening was 1760. Ligands were energy minimized using Open Babel. The RdRp and 3CLpro protein sequences were retrieved from the NCBI database. For Homology Modeling predictions, we used the Swiss model server. Their structure was then energetically minimized using SPDB viewer software and visualized in the CHIMERA UCSF software. Molecular dockings were performed using AutoDock Vina, and candidate drugs were selected based on binding affinity (∆G). Hydrogen bonding and hydrophobic interactions between ligands and proteins were visualized using Ligplot and the Discovery Studio Visualizer v3.0 software. Our results showed 58 drugs against RdRp, which had binding energy of - 8.5 or less, and 69 drugs to inhibit the 3CLpro enzyme with a binding energy of - 8.1 or less. Six drugs based on binding energy and number of hydrogen bonds were chosen for the next step of molecular dynamics (MD) simulations to investigate drug-protein interactions (including Nilotinib, Imatinib and dihydroergotamine for 3clpro and Lapatinib, Dexasone and Relategravir for RdRp). Except for Lapatinib, other drugs-complexes were stable during MD simulation. Raltegravir, an anti-HIV drug, was observed to be the best compound against RdRp based on docking binding energy (-9.5 kcal/mole) and MD results. According to the MD results and binding energy, dihydroergotamine is a suitable candidate for 3clpro inhibition (-9.6 kcal/mol). These drugs were classified into several categories, including antiviral, antibacterial, anti-inflammatory, anti-allergic, cardiovascular, anticoagulant, BPH and impotence, antipsychotic, antimigraine, anticancer, and so on. The common prescription-indications for some of these medication categories appeared somewhat in line with manifestations of COVID-19. We hope that they can be beneficial for patients with certain specific symptoms of SARS-CoV-2 infection, but they can also probably inhibit viral enzymes. We recommend further experimental evaluations in vitro and in vivo on these FDA-approved drugs to assess their potential antiviral effect on SARS-CoV-2.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Reposicionamento de Medicamentos , Inibidores Enzimáticos/uso terapêutico , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/efeitos adversos , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Di-Hidroergotamina/uso terapêutico , Aprovação de Drogas , Interações Hospedeiro-Patógeno , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/metabolismo , Raltegravir Potássico/uso terapêutico , SARS-CoV-2/enzimologia , Estados Unidos , United States Food and Drug Administration
9.
Eur J Med Chem ; 223: 113622, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34147744

RESUMO

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic coronavirus disease (COVID-19), but no specific antiviral drug has been proven effective for controlling this pandemic to date. In this study, several 2-((indol-3-yl)thio)-N-benzyl-acetamides were identified as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors. After a two-round optimization, a new series of 2-((indol-3-yl)thio)-N-benzyl-acetamides was designed, synthesized, and evaluated for SARS-CoV-2 RdRp inhibitory effect. Compounds 6b2, 6b5, 6c9, 6d2, and 6d5 were identified as potent inhibitors with IC50 values of 3.35 ± 0.21 µM, 4.55 ± 0.2 µM, 1.65 ± 0.05 µM, 3.76 ± 0.79 µM, and 1.11 ± 0.05 µM, respectively; the IC50 of remdesivir (control) was measured as 1.19 ± 0.36 µM. All of the compounds inhibited RNA synthesis by SARS-CoV-2 RdRp. The most potent compound 6d5, which showed a stronger inhibitory activity against the human coronavirus HCoV-OC43 than remdesivir, is a promising candidate for further investigation.


Assuntos
Acetamidas/síntese química , Antivirais/síntese química , COVID-19/tratamento farmacológico , Inibidores Enzimáticos/síntese química , RNA Viral/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Acetamidas/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/normas , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/normas , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , SARS-CoV-2/genética , Relação Estrutura-Atividade
10.
Comput Biol Med ; 135: 104555, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34144270

RESUMO

BACKGROUND: Non-structural protein 1 (Nsp1), a virulence agent of SARS-CoV-2, has emerged as an important target for drug discovery. Nsp1 shuts down the host gene function by associating with the 40S ribosomal subunit. METHODS: Molecular interactions, drug-likeness, physiochemical property predictions, and robust molecular dynamics (MD) simulations were employed to discover novel Nsp1 inhibitors. In this study, we evaluated a series of molecules based on the plant (Cedrus deodara) derived α,ß,γ-Himachalenes scaffolds. RESULTS: The results obtained from estimated affinity and ligand efficiency suggested that BCH10, BCH15, BCH16, and BCH17 could act as potential inhibitors of Nsp1. Moreover, MD simulations comprising various MD driven time-dependent analyses and thermodynamic free energy calculations also suggested stable protein-ligand complexes and strong interactions with the binding site. Furthermore, the selected molecules passed drug likeliness parameters and the physiochemical property analysis showed acceptable bioactivity scores. CONCLUSION: The structural parameters of dynamic simulations revealed that the reported molecules could act as lead compounds against SARS-CoV-2 Nsp1 protein.


Assuntos
Cedrus/química , Compostos Fitoquímicos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2 , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , RNA Polimerase Dependente de RNA/química , Subunidades Ribossômicas Menores de Eucariotos , Proteínas não Estruturais Virais/química
11.
Am J Physiol Endocrinol Metab ; 321(2): E246-E251, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34181461

RESUMO

Vitamin D deficiency significantly correlates with the severity of SARS-CoV-2 infection. Molecular docking-based virtual screening studies predict that novel vitamin D and related lumisterol hydroxymetabolites are able to bind to the active sites of two SARS-CoV-2 transcription machinery enzymes with high affinity. These enzymes are the main protease (Mpro) and RNA-dependent RNA polymerase (RdRP), which play important roles in viral replication and establishing infection. Based on predicted binding affinities and specific interactions, we identified 10 vitamin D3 (D3) and lumisterol (L3) analogs as likely binding partners of SARS-CoV-2 Mpro and RdRP and, therefore, tested their ability to inhibit these enzymes. Activity measurements demonstrated that 25(OH)L3, 24(OH)L3, and 20(OH)7DHC are the most effective of the hydroxymetabolites tested at inhibiting the activity of SARS-CoV-2 Mpro causing 10%-19% inhibition. These same derivatives as well as other hydroxylumisterols and hydroxyvitamin D3 metabolites inhibited RdRP by 50%-60%. Thus, inhibition of these enzymes by vitamin D and lumisterol metabolites may provide a novel approach to hindering the SARS-CoV-2 infection.NEW & NOTEWORTHY Active forms of vitamin D and lumisterol can inhibit SARS-CoV-2 replication machinery enzymes, which indicates that novel vitamin D and lumisterol metabolites are candidates for antiviral drug research.


Assuntos
Antivirais/farmacologia , Ergosterol/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Vitamina D/farmacologia , Antivirais/química , Ergosterol/análogos & derivados , Ergosterol/química , Ergosterol/farmacologia , Simulação de Acoplamento Molecular , RNA Polimerase Dependente de RNA/química , SARS-CoV-2/fisiologia , Vitamina D/química
12.
ACS Infect Dis ; 7(6): 1535-1544, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34038639

RESUMO

Coronavirus disease 2019 (COVID-19) is a fatal respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of potential drugs is urgently needed to control the pandemic. RNA dependent RNA polymerase (RdRp) is a conserved protein within RNA viruses and plays a crucial role in the viral life cycle, thus making it an attractive target for development of antiviral drugs. In this study, 101 quinoline and quinazoline derivatives were screened against SARS-CoV-2 RdRp using a cell-based assay. Three compounds I-13e, I-13h, and I-13i exhibit remarkable potency in inhibiting RNA synthesis driven by SARS-CoV-2 RdRp and relatively low cytotoxicity. Among these three compounds, I-13e showed the strongest inhibition upon RNA synthesis driven by SARS-CoV-2 RdRp, the resistance to viral exoribonuclease activity and the inhibitory effect on the replication of CoV, thus holding potential of being drug candidate for treatment of SARS-CoV-2.


Assuntos
Quinazolinas , Quinolinas , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Humanos , Quinazolinas/farmacologia , Quinolinas/farmacologia , RNA Viral/biossíntese
13.
Eur J Med Chem ; 221: 113494, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-33962311

RESUMO

In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC50 = 19.5 µM) and anti-IAV activity (EC50 = 16 µM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47 µM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents.


Assuntos
Antivirais/farmacologia , Multimerização Proteica/efeitos dos fármacos , Pirimidinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Chlorocebus aethiops , Cães , Desenho de Fármacos , Células HEK293 , Humanos , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Testes de Sensibilidade Microbiana , Pirimidinas/síntese química , SARS-CoV-2/efeitos dos fármacos , Triazóis/síntese química , Células Vero
14.
PLoS One ; 16(5): e0251801, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33984041

RESUMO

Drug repurposing studies targeting inhibition of RNA dependent RNA polymerase (RdRP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have exhibited the potential effect of small molecules. In the present work a detailed interaction study between the phytochemicals from Indian medicinal plants and the RdRP of SARS-CoV-2 has been performed. The top four phytochemicals obtained through molecular docking were, swertiapuniside, cordifolide A, sitoindoside IX, and amarogentin belonging to Swertia chirayita, Tinospora cordifolia and Withania somnifera. These ligands bound to the RdRP were further studied using molecular dynamics simulations. The principal component analysis of these systems showed significant conformational changes in the finger and thumb subdomain of the RdRP. Hydrogen bonding, salt-bridge and water mediated interactions supported by MM-GBSA free energy of binding revealed strong binding of cordifolide A and sitoindoside IX to RdRP. The ligand-interacting residues belonged to either of the seven conserved motifs of the RdRP. These residues were polar and charged amino acids, namely, ARG 553, ARG 555, ASP 618, ASP 760, ASP 761, GLU 811, and SER 814. The glycosidic moieties of the phytochemicals were observed to form favourable interactions with these residues. Hence, these phytochemicals may hold the potential to act as RdRP inhibitors owing to their stability in binding to the druggable site.


Assuntos
COVID-19/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Compostos Fitoquímicos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/enzimologia , Antivirais/química , Antivirais/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Fitoquímicos/química , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos
15.
Molecules ; 26(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946996

RESUMO

Respiratory syncytial virus (RSV) is a major pathogen that causes severe lower respiratory tract infection in infants, the elderly and the immunocompromised worldwide. At present no approved specific drugs or vaccines are available to treat this pathogen. Recently, several promising candidates targeting RSV entry and multiplication steps are under investigation. However, it is possible to lead to drug resistance under the long-term treatment. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we tested in vitro two-drug combinations of fusion inhibitors (GS5806, Ziresovir and BMS433771) and RNA-dependent RNA polymerase complex (RdRp) inhibitors (ALS8176, RSV604, and Cyclopamine). The statistical program MacSynergy II was employed to determine synergism, additivity or antagonism between drugs. From the result, we found that combinations of ALS8176 and Ziresovir or GS5806 exhibit additive effects against RSV in vitro, with interaction volume of 50 µM2% and 31 µM2% at 95% confidence interval, respectively. On the other hand, all combinations between fusion inhibitors showed antagonistic effects against RSV in vitro, with volume of antagonism ranging from -50 µM2 % to -176 µM2 % at 95% confidence interval. Over all, our results suggest the potentially therapeutic combinations in combating RSV in vitro could be considered for further animal and clinical evaluations.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Antivirais/química , Antivirais/uso terapêutico , Descoberta de Drogas/métodos , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Quinazolinas/química , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Bibliotecas de Moléculas Pequenas , Tiazepinas/química , Tiazepinas/farmacologia , Tiazepinas/uso terapêutico , Inibidores de Proteínas Virais de Fusão/química , Inibidores de Proteínas Virais de Fusão/farmacologia , Inibidores de Proteínas Virais de Fusão/uso terapêutico
16.
Antiviral Res ; 190: 105078, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33894278

RESUMO

Antiviral therapeutics is one effective avenue to control and end this devastating COVID-19 pandemic. The viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 has been recognized as a valuable target of antivirals. However, the cell-free SARS-CoV-2 RdRp biochemical assay requires the conversion of nucleotide prodrugs into the active triphosphate forms, which regularly occurs in cells yet is a complicated multiple-step chemical process in vitro, and thus hinders the utility of this cell-free assay in the rapid discovery of RdRp inhibitors. In addition, SARS-CoV-2 exoribonuclease provides the proof-reading capacity to viral RdRp, thus creates relatively high resistance threshold of viral RdRp to nucleotide analog inhibitors, which must be examined and evaluated in the development of this class of antivirals. Here, we report a cell-based assay to evaluate the efficacy of nucleotide analog compounds against SARS-CoV-2 RdRp and assess their tolerance to viral exoribonuclease-mediated proof-reading. By testing seven commonly used nucleotide analog viral polymerase inhibitors, Remdesivir, Molnupiravir, Ribavirin, Favipiravir, Penciclovir, Entecavir and Tenofovir, we found that both Molnupiravir and Remdesivir showed the strong inhibition of SARS-CoV-2 RdRp, with EC50 value of 0.22 µM and 0.67 µM, respectively. Moreover, our results suggested that exoribonuclease nsp14 increases resistance of SARS-CoV-2 RdRp to nucleotide analog inhibitors. We also determined that Remdesivir presented the highest resistance to viral exoribonuclease activity in cells. Therefore, we have developed a cell-based SARS-CoV-2 RdRp assay which can be deployed to discover SARS-CoV-2 RdRp inhibitors that are urgently needed to treat COVID-19 patients.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Descoberta de Drogas , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Células A549 , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , COVID-19/virologia , Sobrevivência Celular/efeitos dos fármacos , Exorribonucleases/antagonistas & inibidores , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , RNA Viral/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/antagonistas & inibidores
17.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800884

RESUMO

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.


Assuntos
Substituição de Aminoácidos , Antivirais/farmacologia , Mutação Puntual , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Sofosbuvir/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Zika virus/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Sítios de Ligação , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas/patologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , RNA Viral/biossíntese , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Seleção Genética , Sofosbuvir/uso terapêutico , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Zika virus/enzimologia , Zika virus/genética
18.
J Biol Chem ; 296: 100486, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33647314

RESUMO

Baloxavir marboxil (BXM) is an FDA-approved antiviral prodrug for the treatment of influenza A and B infection and postexposure prophylaxis. The active form, baloxavir acid (BXA), targets the cap-snatching endonuclease (PA) of the influenza virus polymerase complex. The nuclease activity delivers the primer for transcription, and previous reports have shown that BXA blocks the nuclease activity with high potency. However, biochemical studies on the mechanism of action are lacking. Structural data have shown that BXA chelates the two divalent metal ions at the active site, like inhibitors of the human immunodeficiency virus type 1 (HIV-1) integrase or ribonuclease (RNase) H. Here we studied the mechanisms underlying the high potency of BXA and how the I38T mutation confers resistance to the drug. Enzyme kinetics with the recombinant heterotrimeric enzyme (FluB-ht) revealed characteristics of a tight binding inhibitor. The apparent inhibitor constant (Kiapp) is 12 nM, while the I38T mutation increased Kiapp by ∼18-fold. Order-of-addition experiments show that a preformed complex of FluB-ht, Mg2+ ions and BXA is required to observe inhibition, which is consistent with active site binding. Conversely, a preformed complex of FluB-ht and RNA substrate prevents BXA from accessing the active site. Unlike integrase inhibitors that interact with the DNA substrate, BXA behaves like RNase H inhibitors that compete with the nucleic acid at the active site. The collective data support the conclusion that BXA is a tight binding inhibitor and the I38T mutation diminishes these properties.


Assuntos
Dibenzotiepinas/farmacologia , Endonucleases/antagonistas & inibidores , Vírus da Influenza B/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Morfolinas/farmacologia , Piridonas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Triazinas/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Domínio Catalítico , Endonucleases/metabolismo , Humanos , Vírus da Influenza B/enzimologia , Vírus da Influenza B/isolamento & purificação , Influenza Humana/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo
19.
Molecules ; 26(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669720

RESUMO

Coronavirus desease 2019 (COVID-19) is responsible for more than 1.80 M deaths worldwide. A Quantitative Structure-Activity Relationships (QSAR) model is developed based on experimental pIC50 values reported for a structurally diverse dataset. A robust model with only five descriptors is found, with values of R2 = 0.897, Q2LOO = 0.854, and Q2ext = 0.876 and complying with all the parameters established in the validation Tropsha's test. The analysis of the applicability domain (AD) reveals coverage of about 90% for the external test set. Docking and molecular dynamic analysis are performed on the three most relevant biological targets for SARS-CoV-2: main protease, papain-like protease, and RNA-dependent RNA polymerase. A screening of the DrugBank database is executed, predicting the pIC50 value of 6664 drugs, which are IN the AD of the model (coverage = 79%). Fifty-seven possible potent anti-COVID-19 candidates with pIC50 values > 6.6 are identified, and based on a pharmacophore modelling analysis, four compounds of this set can be suggested as potent candidates to be potential inhibitors of SARS-CoV-2. Finally, the biological activity of the compounds was related to the frontier molecular orbitals shapes.


Assuntos
Antivirais/química , COVID-19/enzimologia , Proteases 3C de Coronavírus , Inibidores de Cisteína Proteinase/química , Bases de Dados de Compostos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA , SARS-CoV-2/enzimologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Inibidores de Cisteína Proteinase/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Relação Quantitativa Estrutura-Atividade , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química
20.
Int J Biol Macromol ; 181: 605-611, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33766591

RESUMO

The outbreaks of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in 2019, have highlighted the concerns about the lack of potential vaccines or antivirals approved for inhibition of CoVs infection. SARS-CoV-2 RNA dependent RNA polymerase (RdRp) which is almost preserved across different viral species can be a potential target for development of antiviral drugs, including nucleoside analogues (NA). However, ExoN proofreading activity of CoVs leads to their protection from several NAs. Therefore, potential platforms based on the development of efficient NAs with broad-spectrum efficacy against human CoVs should be explored. This study was then aimed to present an overview on the development of NAs-based drug repurposing for targeting SARS-CoV-2 RdRp by computational analysis. Afterwards, the clinical development of some NAs including Favipiravir, Sofosbuvir, Ribavirin, Tenofovir, and Remdesivir as potential inhibitors of RdRp, were surveyed. Overall, exploring broad-spectrum NAs as promising inhibitors of RdRp may provide useful information about the identification of potential antiviral repurposed drugs against SARS-CoV-2.


Assuntos
COVID-19/tratamento farmacológico , Nucleosídeos/farmacologia , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Antivirais/farmacologia , COVID-19/virologia , Biologia Computacional/métodos , Reposicionamento de Medicamentos/métodos , Humanos , Modelos Moleculares , RNA Polimerase Dependente de RNA/antagonistas & inibidores
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