RESUMEN
Emerging viruses including Nipah, Hendra, Lujo, and Junin viruses have enormous potential to spread rapidly. Nipah virus, after emerging as a zoonosis, has also evolved the capacity for human-to-human transmission. Most of the diseases caused by these pathogens are untreatable and require high biocontainment conditions. Universal methods for rapidly identifying and screening candidate antivirals are urgently needed. We have developed a modular antiviral platform strategy that relies on simple bioinformatic and genetic information about each pathogen. Central to this platform is the use of envelope glycoprotein cDNAs to establish multi-cycle replication systems under BSL2 conditions for viral pathogens that normally require BSL3 and BSL4 facilities. We generated monoclonal antibodies against Nipah G by cDNA immunization in rats, and we showed that these antibodies neutralize both Nipah and Hendra live viruses. We then used these effective Henipavirus inhibitors to validate our screening strategy. Our proposed strategy should contribute to the response capability for emerging infectious diseases, providing a way to initiate antiviral development immediately upon identifying novel viruses.
Asunto(s)
Biología Computacional/métodos , Virus Hendra/metabolismo , Virus Nipah/metabolismo , Animales , Anticuerpos Neutralizantes/química , Antivirales/farmacología , Chlorocebus aethiops , ADN Complementario/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Células HEK293 , Humanos , Inmunoterapia/métodos , Ratas , Tecnología Farmacéutica/métodos , Células Vero , Replicación ViralRESUMEN
Phenotypic chemogenomics studies require screening strategies that account for the complex nature of the experimental system. Unknown mechanism of action and high frequency of false positives and false negatives necessitate iterative experiments based on hypotheses formed on the basis of results from the previous step. Process-driven High Throughput Screening (HTS), aiming to "industrialize" lead finding and developed to maximize throughput, is rarely affording sufficient flexibility to design hypothesis-based experiments.In this contribution, we describe a High Throughput Cherry Picking (HTCP) system based on acoustic dispensing technology that was developed to support a new screening paradigm. We demonstrate the power of hypothesis-based screening in three chemogenomics studies that were recently conducted.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Acústica/instrumentación , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/instrumentación , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Modelos Biológicos , Biología Molecular/métodos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Interfaz Usuario-ComputadorRESUMEN
The special ergoline 1 is a highly potent, selective antagonist of the chemokine receptor CXCR3. The surprising selectivity of this LSD-related compound can be explained by different electronic and steric properties of the ergoline core structure caused by the urea portion of the molecule. Discovery, biopharmaceutical properties and first derivatives of this promising lead compound are discussed.
Asunto(s)
Ergolinas/química , Receptores CXCR3/antagonistas & inhibidores , Animales , Perros , Descubrimiento de Drogas , Ergolinas/farmacología , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Conformación Molecular , Ratas , Receptores CXCR3/metabolismo , Relación Estructura-ActividadRESUMEN
Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 microM in vitro (50% inhibitory concentration, 2 microM), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine's antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.
Asunto(s)
Antivirales/farmacología , Cloroquina/farmacología , Descubrimiento de Drogas/métodos , Virus Hendra/efectos de los fármacos , Virus Nipah/efectos de los fármacos , Animales , Chlorocebus aethiops , Virus Hendra/fisiología , Infecciones por Henipavirus/tratamiento farmacológico , Humanos , Virus Nipah/fisiología , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Replicación ViralRESUMEN
The interaction of the chemokine receptor CXCR4 with its ligand CXCL12 is involved in many biological processes such as hematopoesis, migration of immune cells, as well as in cancer metastasis. CXCR4 also mediates the infection of T-cells with X4-tropic HIV functioning as a coreceptor for the viral envelope protein gp120. Here, we describe highly potent, selective CXCR4 inhibitors that block CXCR4/CXCL12 interactions in vitro and in vivo as well as the infection of target cells by X4-tropic HIV.
Asunto(s)
Receptores CXCR4/química , Tiourea/química , Administración Oral , Animales , Disponibilidad Biológica , Química Farmacéutica/métodos , Quimiocina CXCL12/química , Quimiocinas/metabolismo , Diseño de Fármacos , Proteína gp120 de Envoltorio del VIH/química , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Ratas , Receptores CXCR4/antagonistas & inhibidores , Linfocitos T/metabolismo , Linfocitos T/virologíaRESUMEN
Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.