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 (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
A rapid drug discovery response to influenza outbreaks with the potential to reach pandemic status could help minimize the virus's impact by reducing the time to identify anti-influenza drugs. Although several anti-influenza strategies have been considered in the search for new drugs, only a few therapeutic agents are approved for clinical use. The cytopathic effect induced by the influenza virus in Madin Darby canine kidney (MDCK) cells has been widely used for high-throughput anti-influenza drug screening, but the fact that the MDCK cells are not human cells constitutes a disadvantage when searching for new therapeutic agents for human use. We have developed a highly sensitive cell-based imaging assay for the identification of inhibitors of influenza A and B virus that is high-throughput compatible using the A549 human cell line. The assay has also been optimized for the assessment of the neutralizing effect of anti-influenza antibodies in the absence of trypsin, which allows testing of purified antibodies and serum samples. This assay platform can be applied to full high-throughput screening campaigns or later stages requiring quantitative potency determinations for structure-activity relationships.
Assuntos
Influenza Humana , Animais , Cães , Humanos , Influenza Humana/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Linhagem Celular , Células Madin Darby de Rim Canino , ImunofluorescênciaRESUMO
Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.
Assuntos
COVID-19 , Ensaios de Triagem em Larga Escala , Pandemias , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Antivirais/uso terapêutico , Antivirais/farmacologiaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that has been declared by the World Health Organization as a "priority 1 critical pathogen" needing immediate new strategies for chemotherapy. During infection, P. aeruginosa uses redundant mechanisms to acquire ferric, heme (Hm), or ferrous iron from the host to survive and colonize. Significant efforts have been undertaken to develop siderophore blockers to inhibit ferric iron acquisition by P. aeruginosa, but there is a lack of inhibitors that can block Hm or ferrous iron acquisition by P. aeruginosa. We developed and employed a targeted high-throughput screen (HTS) and identified a molecule(s) that can specifically inhibit the Hm and ferrous iron acquisition systems of P. aeruginosa. Our targeted approach relies on screening a small-molecule library against P. aeruginosa under three growth conditions, where the only variable was the iron source (ferric, Hm, or ferrous iron). Each condition served as a counterscreen for the other, and we identified molecules that inhibit the growth of P. aeruginosa in the presence of only Hm or ferrous iron. Our data indicate that econazole, bithionate, and raloxifene inhibit the growth of P. aeruginosa in the presence of Hm and that oxyquinoline inhibits the growth of P. aeruginosa in the presence of ferrous iron. These iron-specific inhibitors do not interfere with the activity of meropenem, a commercial antipseudomonal, and can also increase meropenem activity. In conclusion, we present a proof of concept of a successful targeted conditional screening method by which we can identify specific iron acquisition inhibitors. This approach is highly adaptable and can easily be extended to any other pathogen. IMPORTANCE Since acquiring iron is paramount to P. aeruginosa's survival and colonization in the human host, developing novel strategies to block the access of P. aeruginosa to host iron will allow us to starve it of an essential nutrient. P. aeruginosa uses siderophore, heme, or ferrous iron uptake systems to acquire iron in the human host. We have developed a novel approach through which we can directly identify molecules that can prevent P. aeruginosa from utilizing heme or ferrous iron. This approach overcomes the need for the in silico design of molecules and identifies structurally diverse biologically active inhibitor molecules. This screening approach is adaptable and can be extended to any pathogen. Since Gram-negative pathogens share many similarities in iron acquisition at both the mechanistic and molecular levels, our screening approach presents a significant opportunity to develop novel broad-spectrum iron acquisition inhibitors of Gram-negative pathogens.
Assuntos
Pseudomonas aeruginosa , Sideróforos , Proteínas de Bactérias , Econazol , Heme , Ferro , Meropeném , Oxiquinolina , Cloridrato de RaloxifenoRESUMO
Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.
Assuntos
NF-kappa B/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , NF-kappa B/agonistas , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/isolamento & purificação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Most of the components of the membrane and protein traffic machinery were discovered by perturbing their functions, either with bioactive compounds or by mutations. However, the mechanisms responsible for exocytic transport vesicle formation at the Golgi and endosomes are still largely unknown. Both the exocytic traffic routes and the signaling pathways that regulate these routes are highly complex and robust, so that defects can be overcome by alternate pathways or mechanisms. A classical yeast genetic screen designed to account for the robustness of the exocytic pathway identified a novel conserved gene, AVL9, which functions in late exocytic transport. We now describe a chemical-genetic version of the mutant screen, in which we performed a high-throughput phenotypic screen of a large compound library and identified novel small-molecule secretory inhibitors. To maximize the number and diversity of our hits, the screen was performed in a pdr5Delta snq2Delta mutant background, which lacks two transporters responsible for pleiotropic drug resistance. However, we found that deletion of both transporters reduced the fitness of our screen strain, whereas the pdr5Delta mutation had a relatively small effect on growth and was also the more important transporter mutation for conferring sensitivity to our hits. In this and similar chemical-genetic yeast screens, using just a single pump mutation might be sufficient for increasing hit diversity while minimizing the physiological effects of transporter mutations.
Assuntos
Exocitose/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Endossomos/metabolismo , Ensaios de Triagem em Larga Escala , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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 VeroRESUMO
The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte's Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research's HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens.
Assuntos
Tecnologia Biomédica/métodos , Estudos de Associação Genética/métodos , Ensaios de Triagem em Larga Escala/métodos , Interferência de RNA , Acústica , SoluçõesRESUMO
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.
Assuntos
Antivirais/química , Antivirais/farmacologia , Benzamidas/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Piperazinas/farmacologia , Animais , Benzamidas/química , Avaliação Pré-Clínica de Medicamentos/métodos , Encefalomielite Equina Venezuelana/tratamento farmacológico , Compostos Heterocíclicos com 2 Anéis/química , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Piperazinas/química , Quinazolinonas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z' factor, which incorporates both standard deviation and signal window. A Z' factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z' factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z' factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.
Assuntos
Testes Genéticos/métodos , Ensaios de Triagem em Larga Escala , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Testes Genéticos/instrumentação , Células HEK293 , Humanos , Técnicas Analíticas Microfluídicas/normas , Reprodutibilidade dos TestesRESUMO
The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC(50), cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC(50) of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antígenos Transformantes de Poliomavirus/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Fenóis/farmacologia , Animais , Antivirais/farmacologia , Compostos Benzidrílicos , Linhagem Celular , Chlorocebus aethiops , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Polyomavirus/enzimologia , Bibliotecas de Moléculas Pequenas/análiseRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.