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 UnidosRESUMO
Ebola virus (EBOV) in body fluids poses risk for virus transmission. However, there are limited experimental data for such matrices on the disinfectant efficacy against EBOV. We evaluated the effectiveness of disinfectants against EBOV in blood on surfaces. Only 5% peracetic acid consistently reduced EBOV titers in dried blood to the assay limit of quantification.
Assuntos
Desinfetantes/farmacologia , Ebolavirus/efeitos dos fármacos , Clareadores/farmacologia , Células Cultivadas/virologia , Teste em Amostras de Sangue Seco , Humanos , Laboratórios , Ácido Peracético/farmacologiaRESUMO
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 (IC50â=â0.84 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 fármacosRESUMO
We sought to determine the mechanisms by which influenza infection of human epithelial cells decreases cystic fibrosis transmembrane conductance regulator (CFTR) expression and function. We infected human bronchial epithelial (NHBE) cells and murine nasal epithelial (MNE) cells with various strains of influenza A virus. Influenza infection significantly reduced CFTR short circuit currents (Isc) and protein levels at 8 hours postinfection. We then infected CFTR expressing human embryonic kidney (HEK)-293 cells (HEK-293 CFTRwt) with influenza virus encoding a green fluorescent protein (GFP) tag and performed whole-cell and cell-attached patch clamp recordings. Forskolin-stimulated, GlyH-101-sensitive CFTR conductances, and CFTR open probabilities were reduced by 80% in GFP-positive cells; Western blots also showed significant reduction in total and plasma membrane CFTR levels. Knockdown of the influenza matrix protein 2 (M2) with siRNA, or inhibition of its activity by amantadine, prevented the decrease in CFTR expression and function. Lysosome inhibition (bafilomycin-A1), but not proteasome inhibition (lactacystin), prevented the reduction in CFTR levels. Western blots of immunoprecipitated CFTR from influenza-infected cells, treated with BafA1, and probed with antibodies against lysine 63-linked (K-63) or lysine 48-linked (K-48) polyubiquitin chains supported lysosomal targeting. These results highlight CFTR damage, leading to early degradation as an important contributing factor to influenza infection-associated ion transport defects.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Proteínas da Matriz Viral/fisiologia , Animais , Apoptose , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Vírus da Influenza A/genética , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Transporte de Íons , Lisossomos/metabolismo , Camundongos , Necrose , Técnicas de Patch-Clamp , Proteólise , Transfecção , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genéticaRESUMO
Death by respiratory complications from influenza infections continues to be a major global health concern. Antiviral drugs are widely available for therapy and prophylaxis, but viral mutations have resulted in resistance that threatens to reduce the long-term utility of approved antivirals. Vaccination is the best method for controlling influenza, but vaccine strategies are blunted by virus antigenic drift and shift. Genetic shift in particular has led to four pandemics in the last century, which have prompted the development of efficient global surveillance and vaccination programs. Although the influenza pandemic of 2009 emphasized the need for the rapid standardization of global surveillance methods and the preparation and dissemination of global assay standards for improved reporting and diagnostic tools, outbreaks of novel influenza strains continue to occur, and current efforts must be enhanced by aggressive public education programs to promote increased vaccination rates in the global population. Recently, a novel H7N9 avian influenza virus with potential to become a pandemic strain emerged in China and was transmitted from animals to humans with a demonstrated >20% mortality rate. Sporadic outbreaks of highly lethal avian virus strains have already increased public awareness and altered annual vaccine production strategies to prevent the natural adaption of this virus to human-to-human transmission. Additional strategies for combating influenza include advancement of new antivirals for unexploited viral or host cellular targets; novel adjuvants and alternate vaccine delivery systems; and development of universal protein, DNA, or multivalent vaccines designed to increase immune responsiveness and enhance public health response times.
Assuntos
Antivirais/uso terapêutico , Controle de Doenças Transmissíveis/métodos , Deriva Genética , Vacinas contra Influenza , Influenza Humana , Pandemias , Controle de Doenças Transmissíveis/organização & administração , Humanos , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/mortalidade , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Educação de Pacientes como Assunto/métodosRESUMO
The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Canais Iônicos/fisiologia , Proteínas da Matriz Viral/farmacologia , Amantadina/farmacologia , Animais , Benzoatos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Furanos/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Canais Iônicos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Pirazóis/farmacologia , Via Secretória/efeitos dos fármacos , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , XenopusRESUMO
Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.
Assuntos
Antivirais/farmacologia , Vaccinia virus/efeitos dos fármacos , Proteínas Virais/metabolismo , Linhagem Celular , DNA Glicosilases/metabolismo , Humanos , Ligação ProteicaRESUMO
The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein and an important virulence factor. It is composed of two well-characterized domains linked by a short, but not well crystallographically defined, region of unknown function. To study the possible function of this region, we introduced alanine substitutions to replace the two highly conserved leucine residues at amino acid positions 69 and 77. The mutant L69,77A NS1 protein retained wild-type (WT)-comparable binding capabilities to dsRNA, cleavage and polyadenylation specificity factor 30 and the p85ß subunit of PI3K. A mutant influenza A virus expressing the L69,77A NS1 protein was generated using reverse genetics. L69,77A NS1 virus infection induced significantly higher levels of beta interferon (IFN-ß) expression in Madin-Darby canine kidney (MDCK) cells compared with WT NS1 virus. In addition, the replication rate of the L69,77A NS1 virus was substantially lower in MDCK cells but not in Vero cells compared with the WT virus, suggesting that the L69,77A NS1 protein does not fully antagonize IFN during viral replication. L69,77A NS1 virus infection was not able to activate the PI3K/Akt anti-apoptotic pathway, suggesting that the mutant NS1 protein may not be localized such that it has access to p85ß in vivo during infection, which was supported by the altered subcellular localization pattern of the mutant NS1 compared with WT NS1 after transfection or virus infection. Our data demonstrate that this linker region between the two domains is critical for the functions of the NS1 protein during influenza A virus infection, possibly by determining the protein's correct subcellular localization.
Assuntos
Substituição de Aminoácidos , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Espaço Intracelular/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Alanina/química , Alanina/genética , Alanina/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Cães , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/imunologia , Interferon beta/genética , Interferon beta/imunologia , Transporte Proteico , Proteínas não Estruturais Virais/genéticaRESUMO
We describe a primary high-throughput screen that uses the reporter strain Bacillus subtilis BAU-102 to identify antibiotics that induce autolysis. The screen measures autolysis in terms of the incipient release of recombinant Escherichia coli beta-galactosidase (beta-Gal) from the periplasmic space of B. subtilis owing to a loss of integrity of the cell wall. In a model screen, beta-Gal release values for 79 members of a library consisting of antibiotics and related compounds were collected, sorted, and plotted as a function of rank. Inducers of autolysis, which included compounds that inhibit cell wall synthesis and those that do not, were readily differentiated from other members of the library on the basis of their elevated beta-galactosidase release responses. The results of the BAU-102 model screen called attention to the antibacterial activity of drugs normally used in other applications, describable as "repurposed." Thus, the screen independently identified the potential antibacterial properties of the antifungal drug miconazole and of the antileishmaniasis drug miltefosine. Daptomycin-induced release of beta-Gal was also detected and occurred in a Ca(2+)-dependent manner.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacteriólise/efeitos dos fármacos , Bacillus subtilis/metabolismo , Daptomicina/farmacologia , Miconazol/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , beta-Galactosidase/metabolismoRESUMO
The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride-sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride-sensitive currents in both Xenopus oocytes, injected with human alpha-, beta-, and gamma-ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10-aa region within the M2 C terminus prevented 70% of this effect. The M2 ENaC down-regulation occurred at normal pH and was prevented by MG-132, a proteasome and lysosome inhibitor. M2 had no effect on Liddle ENaCs, which have decreased affinity for Nedd4-2. H441 and A549 cells transfected with M2 showed higher levels of reactive oxygen species, as shown by the activation of redox-sensitive dyes. Pretreatment with glutathione ester, which increases intracellular reduced thiol concentrations, or protein kinase C (PKC) inhibitors prevented the deleterious effects of M2 on ENaCs. The data suggest that M2 protein increases steady-state concentrations of reactive oxygen intermediates that simulate PKC and decrease ENaCs by enhancing endocytosis and its subsequent destruction by the proteasome. These novel findings suggest a mechanism for the influenza-induced rhinorrhea and life-threatening alveolar edema in humans.
Assuntos
Bloqueadores do Canal de Sódio Epitelial , Espécies Reativas de Oxigênio/metabolismo , Proteínas da Matriz Viral/fisiologia , Amilorida/farmacologia , Animais , Células Cultivadas , Humanos , Oócitos/metabolismo , Proteína Quinase C/metabolismo , Transfecção , Xenopus laevisRESUMO
The spread of highly pathogenic avian influenza across geographical and species barriers underscores the increasing need for novel antivirals to compliment vaccination and existing antiviral therapies. Identification of new antiviral lead compounds depends on robust primary assays for high-throughput screening (HTS) of large compound libraries. We have developed a cell-based screen for potential influenza antivirals that measures the cytopathic effect (CPE) induced by influenza virus (A/Udorn/72, H3N2) infection in Madin Darby canine kidney (MDCK) cells using the luminescent-based CellTiter Glo system. This 72 h assay is validated for HTS in 384-well plates and performs more consistently and reliably than methods using neutral red, with Z values>0.8, signal-to-background>30 and signal-to-noise>10. In a blinded pilot screen (n=10,781) at 10 microM concentration, four compounds (with previously demonstrated efficacy against influenza) inhibited viral-induced CPE by >50%, with EC50/CC50 values comparable to those determined by other cell-based assays, thereby validating this assay accuracy and ability to simultaneously evaluate compound cellular availability and/or toxicity. This assay is translatable for screening against other influenza strains, such as avian flu, and may facilitate identification of antivirals for other viruses that induce CPE, such as West Nile or Dengue.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Luminescência , Animais , Linhagem Celular , Efeito Citopatogênico Viral , Cães , Humanos , Vírus da Influenza A Subtipo H3N2/patogenicidade , Testes de Sensibilidade Microbiana/métodos , Vermelho Neutro , Oseltamivir/farmacologia , Ribavirina/farmacologiaRESUMO
In support of the response to the 2013-2016 Ebola virus disease (EVD) outbreak in Western Africa, we investigated the persistence of Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 (EBOV/Mak-C05) on non-porous surfaces that are representative of hospitals, airplanes, and personal protective equipment. We performed persistence studies in three clinically-relevant human fluid matrices (blood, simulated vomit, and feces), and at environments representative of in-flight airline passenger cabins, environmentally-controlled hospital rooms, and open-air Ebola treatment centers in Western Africa. We also compared the surface stability of EBOV/Mak-C05 to that of the prototype Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Mayinga (EBOV/Yam-May), in a subset of these conditions. We show that on inert, non-porous surfaces, EBOV decay rates are matrix- and environment-dependent. Among the clinically-relevant matrices tested, EBOV persisted longest in dried human blood, had limited viability in dried simulated vomit, and did not persist in feces. EBOV/Mak-C05 and EBOV/Yam-May decay rates in dried matrices were not significantly different. However, during the drying process in human blood, EBOV/Yam-May showed significantly greater loss in viability than EBOV/Mak-C05 under environmental conditions relevant to the outbreak region, and to a lesser extent in conditions relevant to an environmentally-controlled hospital room. This factor may contribute to increased communicability of EBOV/Mak-C05 when surfaces contaminated with dried human blood are the vector and may partially explain the magnitude of the most recent outbreak, compared to prior outbreaks. These EBOV persistence data will improve public health efforts by informing risk assessments, structure remediation decisions, and response procedures for future EVD outbreaks.
Assuntos
Ebolavirus/fisiologia , Equipamento de Proteção Individual/virologia , Animais , Sangue/virologia , Chlorocebus aethiops , Ebolavirus/patogenicidade , Fezes/virologia , Humanos , Umidade , Especificidade da Espécie , Células Vero/virologia , Vômito/virologiaRESUMO
Human respiratory syncytial virus (hRSV) is a highly contagious Paramyxovirus that infects most children by age two, generating an estimated 75,000-125,000 hospitalizations in the U.S. annually. hRSV is the most common cause of bronchiolitis and pneumonia among infants and children under 1year of age, with significant mortality among high-risk groups. A regulatory agency-approved vaccine is not available, and existing prophylaxis and therapies are limited to use in high-risk pediatric patients; thus additional therapies are sorely needed. Here, we identify a series of benzimidazole analogs that inhibit hRSV infection in vitro with high potency, using a previously-reported high-throughput screening assay. The lead compound, SRI 29365 (1-[6-(2-furyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl]methyl-1H-benzimidazole), has an EC50 of 66µM and a selectivity >50. We identified additional compounds with varying potencies by testing commercially-available chemical analogs. Time-of-addition experiments indicated that SRI 29365 effectively inhibits viral replication only if present during the early stages of viral infection. We isolated a virus with resistance to SRI 29365 and identified mutations in the transmembrane domain of the viral G protein genomic sequence that suggested that the compound inhibits G-protein mediated attachment of hRSV to cells. Additional experiments with multiple cell types indicated that SRI 29365 antiviral activity correlates with the binding of cell surface heparin by full-length G protein. Lastly, SRI 29365 did not reduce hRSV titers or morbidity/mortality in efficacy studies using a cotton rat model. Although SRI 29365 and analogs inhibit hRSV replication in vitro, this work suggests that the G-protein may not be a valid drug target in vivo.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas do Envelope Viral/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Modelos Animais de Doenças , Farmacorresistência Viral , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Sigmodontinae , Análise de Sobrevida , Proteínas do Envelope Viral/genéticaRESUMO
High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories.
Assuntos
Bioensaio/instrumentação , Contenção de Riscos Biológicos/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Laboratórios , Tecnologia Farmacêutica/instrumentação , Desenho de Fármacos , Desenho de Equipamento , Análise de Falha de Equipamento , Robótica/instrumentação , Manejo de Espécimes/instrumentaçãoAssuntos
Defesa Civil , Surtos de Doenças/prevenção & controle , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza , Influenza Humana/prevenção & controle , Adolescente , Adulto , Defesa Civil/economia , Análise Custo-Benefício , Surtos de Doenças/economia , Custos de Medicamentos , Custos de Cuidados de Saúde , Humanos , Esquemas de Imunização , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/economia , Vacinas contra Influenza/provisão & distribuição , Influenza Humana/economia , Influenza Humana/mortalidade , Influenza Humana/virologia , Pessoa de Meia-Idade , Estações do Ano , Replicação Viral , Adulto JovemRESUMO
During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus-infected human cells.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/enzimologia , Proteínas Serina-Treonina Quinases/química , RNA Interferente Pequeno/genética , Replicação Viral , Células A549 , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Apoptose , Biotina/análogos & derivados , Biotina/química , Sobrevivência Celular , Cromatografia Líquida , Descoberta de Drogas , Humanos , Espectrometria de Massas , Quinase 1 Relacionada a NIMA/química , Peptídeos/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinase 3 , Carga ViralRESUMO
Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 µM and selectivities >10. Three sulfonamide compounds with EC50 values <12 µM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.
Assuntos
Antivirais/administração & dosagem , Antivirais/química , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Vírus Nipah/efeitos dos fármacos , Vírus Nipah/fisiologia , Sulfonamidas/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Chlorocebus aethiops , Contenção de Riscos Biológicos/instrumentação , Relação Dose-Resposta a Droga , Desenho de Equipamento , Análise de Falha de Equipamento , Robótica/instrumentação , Células Vero , Replicação Viral/fisiologiaRESUMO
A quinazolinedione-derived screening hit 2 was discovered with cellular antiviral activity against respiratory syncytial virus (CPE EC50 = 2.1 µM), moderate efficacy in reducing viral progeny (4.2 log at 10 µM), and marginal cytotoxic liability (selectivity index, SI â¼ 24). Scaffold optimization delivered analogs with improved potency and selectivity profiles. Most notable were compounds 15 and 19 (EC50 = 300-500 nM, CC50 > 50 µM, SI > 100), which significantly reduced viral titer (>400,000-fold), and several analogs were shown to block the activity of the RNA-dependent RNA-polymerase complex of RSV.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Quinazolinonas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Benzamidas/síntese química , Desenho de Fármacos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Quinazolinonas/síntese química , Quinazolinonas/química , RNA Polimerase Dependente de RNA/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Relação Estrutura-AtividadeRESUMO
Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.
Assuntos
Antibacterianos , Cápsulas Bacterianas/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Infecções por Escherichia coli/patologia , Feminino , Humanos , Camundongos , Infecções Urinárias/patologiaRESUMO
Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 = 0.8 µM), limited cytotoxic liability (CC50 > 50 µM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 µM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC50 = 0.02-0.04 µM, CC50 > 50 µM) while limiting in vitro viral replication (EC90 = 0.17 µM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg(-1) day(-1) and viral replication appeared to be inhibited through interference of viral nonstructural proteins.