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1.
Cell ; 161(6): 1252-65, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26046436

RESUMO

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.


Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , National Institutes of Health (U.S.) , Estados Unidos
2.
PLoS Pathog ; 16(5): e1008204, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357162

RESUMO

Zika virus (ZIKV) can infect and cause microcephaly and Zika-associated neurological complications in the developing fetal and adult brains. In terms of pathogenesis, a critical question is how ZIKV overcomes the barriers separating the brain from the circulation and gains access to the central nervous system (CNS). Despite the importance of ZIKV pathogenesis, the route ZIKV utilizes to cross CNS barriers remains unclear. Here we show that in mouse models, ZIKV-infected cells initially appeared in the periventricular regions of the brain, including the choroid plexus and the meninges, prior to infection of the cortex. The appearance of ZIKV in cerebrospinal fluid (CSF) preceded infection of the brain parenchyma. Further the brain infection was significantly attenuated by neutralization of the virus in the CSF, indicating that ZIKV in the CSF at the early stage of infection might be responsible for establishing a lethal infection of the brain. We show that cells infected by ZIKV in the choroid plexus were pericytes. Using in vitro systems, we highlight the possibility that ZIKV crosses the blood-CSF barrier by disrupting the choroid plexus epithelial layer. Taken together, our results suggest that ZIKV might exploit the blood-CSF barrier rather than the blood-brain barrier to invade the CNS.


Assuntos
Plexo Corióideo/patologia , Pericitos/patologia , Infecção por Zika virus/patologia , Animais , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Sistema Nervoso Central/patologia , Chlorocebus aethiops , Plexo Corióideo/metabolismo , Plexo Corióideo/virologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcefalia/complicações , Microcefalia/virologia , Doenças do Sistema Nervoso , Pericitos/metabolismo , Pericitos/virologia , Cultura Primária de Células , Células Vero , Zika virus/fisiologia , Infecção por Zika virus/virologia
3.
J Immunol ; 200(10): 3556-3567, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29610142

RESUMO

Silicosis is a lung inflammatory disease caused by chronic exposure to crystalline silica (CS). Leukotriene B4 (LTB4) plays an important role in neutrophilic inflammation, which drives silicosis and promotes lung cancer. In this study, we examined the mechanisms involved in CS-induced inflammatory pathways. Phagocytosis of CS particles is essential for the production of LTB4 and IL-1ß in mouse macrophages, mast cells, and neutrophils. Phagosomes enclosing CS particles trigger the assembly of lipidosome in the cytoplasm, which is likely the primary source of CS-induced LTB4 production. Activation of the JNK pathway is essential for both CS-induced LTB4 and IL-1ß production. Studies with bafilomycin-A1- and NLRP3-deficient mice revealed that LTB4 synthesis in the lipidosome is independent of inflammasome activation. Small interfering RNA knockdown and confocal microscopy studies showed that GTPases Rab5c, Rab40c along with JNK1 are essential for lipidosome formation and LTB4 production. BI-78D3, a JNK inhibitor, abrogated CS-induced neutrophilic inflammation in vivo in an air pouch model. These results highlight an inflammasome-independent and JNK activation-dependent lipidosome pathway as a regulator of LTB4 synthesis and CS-induced sterile inflammation.


Assuntos
Inflamassomos/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Leucotrieno B4/metabolismo , Dióxido de Silício/farmacologia , Animais , Linhagem Celular , Humanos , Interleucina-1beta/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Células RAW 264.7 , Silicose/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
4.
Antimicrob Agents Chemother ; 60(8): 4552-62, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27185801

RESUMO

Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.


Assuntos
Antivirais/farmacologia , Interferon Tipo I/metabolismo , Pirimidinas/biossíntese , Linhagem Celular , HIV-1/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/efeitos dos fármacos
5.
PLoS Pathog ; 10(6): e1004213, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24967809

RESUMO

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.


Assuntos
Antivirais/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Encefalomielite Equina Venezuelana/tratamento farmacológico , Quinazolinonas/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Farmacorresistência Viral/genética , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos C3H , Especificidade da Espécie , Relação Estrutura-Atividade , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
6.
J Virol ; 87(20): 10997-1007, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23903835

RESUMO

In vitro, ribavirin acts as a lethal mutagen in Hantaan virus (HTNV)-infected Vero E6 cells, resulting in an increased mutation load and viral population extinction. In this study, we asked whether ribavirin treatment in the lethal, suckling mouse model of HTNV infection would act similarly. The HTNV genomic RNA (vRNA) copy number and infectious virus were measured in lungs of untreated and ribavirin-treated mice. In untreated, HTNV-infected mice, the vRNA copy number increased for 10 days postinfection (dpi) and thereafter remained constant through 26 dpi. Surprisingly, in ribavirin-treated, HTNV-infected mice, vRNA levels were similar to those in untreated mice between 10 and 26 dpi. Infectious virus levels, however, were different: in ribavirin-treated mice, the amount of infectious HTNV was significantly decreased relative to that in untreated mice, suggesting that ribavirin reduced the specific infectivity of the virus (amount of infectious virus produced per vRNA copy). Mutational analysis revealed a ribavirin-associated elevation in mutation frequency in HTNV vRNA similar to that previously reported in vitro. Codon-based analyses of rates of nonsynonymous (dN) and synonymous (dS) substitutions in the S segment revealed a positive selection for codons within the HTNV N protein gene in the ribavirin-treated vRNA population. In contrast, the vRNA population in untreated, HTNV-infected mice showed a lower level of diversity, reflecting purifying selection for the wild-type genome. In summary, these experiments show two different evolutionary paths that Hantavirus may take during infection in a lethal murine model of disease, as well as the importance of the in vivo host environment in the evolution of the virus, which was not apparent in our prior in vitro model system.


Assuntos
Antivirais/administração & dosagem , Evolução Molecular , Vírus Hantaan/genética , Febre Hemorrágica com Síndrome Renal/virologia , RNA Viral/genética , Ribavirina/administração & dosagem , Animais , Animais Recém-Nascidos , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Vírus Hantaan/isolamento & purificação , Febre Hemorrágica com Síndrome Renal/tratamento farmacológico , Pulmão/virologia , Camundongos , Camundongos Endogâmicos ICR , Taxa de Mutação , Gravidez , Análise de Sequência de DNA , Carga Viral
7.
Virol J ; 10: 19, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-23302182

RESUMO

BACKGROUND: Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial. RESULTS: We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 µM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9. CONCLUSIONS: A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.


Assuntos
Antivirais/isolamento & purificação , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Automação Laboratorial/métodos , Efeito Citopatogênico Viral/efeitos dos fármacos , Células Hep G2 , Hepatócitos/virologia , Humanos , Medições Luminescentes , Potexvirus
8.
SLAS Discov ; 27(1): 8-19, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35058179

RESUMO

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.


Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Catepsina L/antagonistas & inibidores , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
9.
Molecules ; 15(3): 1690-704, 2010 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-20336008

RESUMO

West Nile virus (WNV) is a positive sense, single-stranded RNA virus that can cause illness in humans when transmitted via mosquito vectors. Unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. We developed a high throughput screen to discover small molecule probes that inhibit virus infection of Vero E6 cells. A primary screen of a 13,001 compound library at a 10 microM final concentration was conducted using the 384-well format. Z' values ranged from 0.54-0.83 with a median of 0.74. Average S/B was 17 and S/N for each plate ranged from 10.8 to 23.9. Twenty-six compounds showed a dose response in the HT screen and were further evaluated in a time of addition assay and in a titer reduction assay. Seven compounds showed potential as small molecule probes directed at WNV. The hit rate from the primary screen was 0.185% (24 compounds out of 13,001 compounds) and from the secondary screens was 0.053% (7 out of 13,001 compounds) respectively.


Assuntos
Antivirais/farmacologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Animais , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Células Vero
10.
Antiviral Res ; 174: 104674, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31816348

RESUMO

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2-4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 µM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.


Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Piperazinas/farmacologia , RNA Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Encefalomielite Equina Venezuelana/tratamento farmacológico , Encefalomielite Equina Venezuelana/virologia , Cavalos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Concentração Inibidora 50 , Rim/citologia , RNA Viral/biossíntese , Células Vero
11.
Antiviral Res ; 167: 25-34, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30970271

RESUMO

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.


Assuntos
Benzamidas , Benzamidinas , Farmacorresistência Viral/genética , Vírus da Encefalite Equina do Leste/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Piperazinas , Animais , Antivirais/síntese química , Antivirais/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Benzamidinas/síntese química , Benzamidinas/farmacologia , Linhagem Celular , Encefalomielite Equina do Leste/tratamento farmacológico , Encefalomielite Equina do Leste/prevenção & controle , Encefalomielite Equina Venezuelana/tratamento farmacológico , Encefalomielite Equina Venezuelana/prevenção & controle , Genes Virais , Camundongos , Mutação , Piperazinas/síntese química , Piperazinas/farmacologia
12.
J Biomol Screen ; 13(9): 879-87, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18812571

RESUMO

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-microM compound concentration against influenza strain A/Udorn/72 (H3N2). The "hit" rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-microM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI(50) = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI(50) > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Vírus da Influenza A Subtipo H3N2/química , Animais , Automação , Linhagem Celular , Química Farmacêutica/métodos , Cães , Desenho de Fármacos , Concentração Inibidora 50 , Modelos Químicos , Ribavirina/farmacologia , Replicação Viral/efeitos dos fármacos
13.
Antiviral Res ; 79(1): 19-27, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18394724

RESUMO

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-beta-ribosides and identified one with antiviral activity, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC(50) value of 10 and 4.4 microM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a approximately 25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1+/-0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5'-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Febre Hemorrágica com Síndrome Renal/tratamento farmacológico , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Orthohantavírus/efeitos dos fármacos , Triazóis/síntese química , Triazóis/farmacologia , Animais , Antivirais/metabolismo , Chlorocebus aethiops , Feminino , Genoma Viral/efeitos dos fármacos , Guanosina/antagonistas & inibidores , Guanosina/metabolismo , Guanosina Trifosfato/antagonistas & inibidores , Guanosina Trifosfato/metabolismo , Orthohantavírus/genética , Orthohantavírus/metabolismo , Febre Hemorrágica com Síndrome Renal/virologia , Humanos , Camundongos , Camundongos Endogâmicos , Mutação/efeitos dos fármacos , Nucleosídeos/metabolismo , Ribavirina/análogos & derivados , Ribavirina/síntese química , Ribavirina/metabolismo , Ribavirina/farmacologia , Triazóis/metabolismo , Células Vero
14.
Antiviral Res ; 138: 47-56, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27919709

RESUMO

Recent studies have clearly underscored the association between Zika virus (ZIKV) and severe neurological diseases such as microcephaly and Guillain-Barre syndrome. Given the historical complacency surrounding this virus, however, no significant antiviral screenings have been performed to specifically target ZIKV. As a result, there is an urgent need for a validated screening method and strategy that is focused on highlighting potential anti-ZIKV inhibitors that can be further advanced via rigorous validation and optimization. To address this critical gap, we sought to test whether a cell-based assay that measures protection from the ZIKV-induced cytopathic effect could serve as a high-throughput screen assay for discovering novel anti-ZIKV inhibitors. Employing this approach, we tested the anti-ZIKV activity of previously known broad-spectrum antiviral compounds and discovered several compounds (e.g., NITD008, SaliPhe, and CID 91632869) with anti-ZIKV activity. Interestingly, while GTP synthesis inhibitors (e.g., ribavirin or mycophenolic acid) were too toxic or showed no anti-ZIKV activity (EC50 > 50 µM), ZIKV was highly susceptible to pyrimidine synthesis inhibitors (e.g., brequinar) in the assay. We amended the assay into a high-throughput screen (HTS)-compatible 384-well format and then screened the NIH Clinical Compound Collection library, which includes a total of 727 compounds organized, using an 8-point dose response format with two Zika virus strains (MR766 and PRVABC59, a recent human isolate). The screen discovered 6-azauridine and finasteride as potential anti-ZIKV inhibitors with EC50 levels of 3.18 and 9.85 µM for MR766, respectively. We further characterized the anti-ZIKV activity of 6-azauridine and several pyrimidine synthesis inhibitors such as brequinar in various secondary assays including an antiviral spectrum test within flaviviruses and alphaviruses, Western blot (protein), real-time PCR (RNA), and plaque reduction assays (progeny virus). From these assays, we discovered that brequinar has potent anti-ZIKV activity. Our results show that a broad anti-ZIKV screen of compound libraries with our CPE-based HTS assay will reveal multiple chemotypes that could be pursued as lead compounds for therapies to treat ZIKV-associated diseases or as molecular probes to study the biology of the ZIKV replication mechanism.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Zika virus/efeitos dos fármacos , Animais , Azauridina/farmacologia , Compostos de Bifenilo/farmacologia , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ribavirina/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacos , Infecção por Zika virus/virologia
15.
Sci Rep ; 7(1): 16652, 2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29192272

RESUMO

We report here that in rat and human neuroprogenitor cells as well as rat embryonic cortical neurons Zika virus (ZIKV) infection leads to ribosomal stress that is characterized by structural disruption of the nucleolus. The anti-nucleolar effects were most pronounced in postmitotic neurons. Moreover, in the latter system, nucleolar presence of ZIKV capsid protein (ZIKV-C) was associated with ribosomal stress and apoptosis. Deletion of 22 C-terminal residues of ZIKV-C prevented nucleolar localization, ribosomal stress and apoptosis. Consistent with a casual relationship between ZIKV-C-induced ribosomal stress and apoptosis, ZIKV-C-overexpressing neurons were protected by loss-of-function manipulations targeting the ribosomal stress effector Tp53 or knockdown of the ribosomal stress mediator RPL11. Finally, capsid protein of Dengue virus, but not West Nile virus, induced ribosomal stress and apoptosis. Thus, anti-nucleolar and pro-apoptotic effects of protein C are flavivirus-species specific. In the case of ZIKV, capsid protein-mediated ribosomal stress may contribute to neuronal death, neurodevelopmental disruption and microcephaly.


Assuntos
Apoptose , Proteínas do Capsídeo/metabolismo , Neurônios/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Proteína Supressora de Tumor p53/metabolismo , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Animais , Proteínas do Capsídeo/genética , Nucléolo Celular/metabolismo , Células Cultivadas , Feminino , Expressão Gênica , Interações Hospedeiro-Patógeno , Neurônios/virologia , Transporte Proteico , Ratos , Infecção por Zika virus/virologia
16.
PLoS One ; 8(2): e56602, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23441208

RESUMO

To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009-2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe disease progression.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Fenótipo , Adulto , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Citocinas/metabolismo , Feminino , Genes Virais , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/imunologia , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , Macrófagos/imunologia , Macrófagos/virologia , Masculino , Camundongos , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/mortalidade , Análise de Componente Principal , Virulência , Replicação Viral , Redução de Peso
17.
J Med Chem ; 55(20): 8582-7, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23043370

RESUMO

A high-throughput, cell-based screen was used to identify chemotypes as inhibitors for human respiratory syncytial virus (hRSV). Optimization of a sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC50 = 2.3 ± 0.8 µM) with marginal cytotoxicity (CC50 = 30.9 ± 1.1 µM) and reduced viral titer by 100-fold. Compared to ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity index.


Assuntos
Antivirais/síntese química , Pirrolidinas/síntese química , Quinolinas/síntese química , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Sulfonamidas/síntese química , Sulfonas/síntese química , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Efeito Citopatogênico Viral/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Pirrolidinas/química , Pirrolidinas/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Vírus Sinciciais Respiratórios/fisiologia , Ribavirina/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonas/química , Sulfonas/farmacologia , Carga Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
18.
Antiviral Res ; 83(1): 80-5, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19501259

RESUMO

As part of an ongoing effort to develop new antiviral nucleoside analogs, our interest was drawn to N(1)-aryl purines as a novel structural class and potential scaffold for drug discovery. Herein, we describe the synthesis of N(1)-3-fluorophenyl-inosine (FPI) and N(1)-3-fluorophenyl-hypoxanthine (FP-Hx) and their antiviral activity against hantaviruses. The EC(50) for FPI and FP-Hx were 94 and 234microM, respectively, against Hantaan virus. FPI was not toxic to mammalian cells at concentrations that exhibited antiviral activity. Analysis of its metabolism revealed a low conversion of FPI in Vero E6 or human cells to a 5'-triphosphate, and it was a poor substrate for human purine nucleoside phosphorylase. Further, the compound did not alter GTP levels indicating FPI does not inhibit inosine monophosphate dehydrogenase. With respect to the virus, FPI did not decrease viral RNA levels or increase the mutation frequency of the viral RNA. This suggests that the antiviral activity of FPI might be solely due to the interaction of FPI or its metabolites with viral or host proteins involved in post-replication events that would affect the levels of infectious virus released. Synthesis of other compounds structurally similar to FPI is warranted to identify more potent agents that selectively abrogate production of infectious virus.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Vírus Hantaan/efeitos dos fármacos , Inosina/análogos & derivados , Animais , Biotransformação , Linhagem Celular , Chlorocebus aethiops , Humanos , Hipoxantinas/síntese química , Hipoxantinas/farmacologia , Hipoxantinas/toxicidade , Concentração Inibidora 50 , Inosina/síntese química , Inosina/farmacologia , Inosina/toxicidade , Testes de Sensibilidade Microbiana
19.
J Virol ; 81(21): 11722-9, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17699579

RESUMO

The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5'-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.


Assuntos
Antivirais/farmacologia , Vírus Hantaan/genética , Vírus Hantaan/metabolismo , Mutação , Ribavirina/farmacologia , Animais , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Frequência do Gene , Genoma Viral , Guanosina Trifosfato/metabolismo , Vírus Hantaan/efeitos dos fármacos , Ácido Micofenólico/metabolismo , Compostos Organosselênicos/farmacologia , RNA Viral/química , Ribavirina/análogos & derivados , Ribonucleosídeos/farmacologia , Células Vero
20.
Antimicrob Agents Chemother ; 51(1): 84-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17060520

RESUMO

Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5'-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5'-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 mug/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 mug/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.


Assuntos
Vírus Hantaan/efeitos dos fármacos , IMP Desidrogenase/metabolismo , Nucleotídeos/metabolismo , Ribavirina/farmacologia , Animais , Antivirais/farmacologia , Chlorocebus aethiops , Guanosina/farmacologia , Guanosina Trifosfato/metabolismo , Vírus Hantaan/genética , Vírus Hantaan/metabolismo , IMP Desidrogenase/antagonistas & inibidores , Ácido Micofenólico/farmacologia , RNA Viral/metabolismo , Fatores de Tempo , Células Vero , Replicação Viral/efeitos dos fármacos
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