Screening for Host Proteins with Pro- and Antiviral Activity Using High-Throughput RNAi

2013 ◽  
pp. 71-90 ◽  
Author(s):  
Samantha J. Griffiths
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
Host Proteins

scholarly journals Asymmetric synthesis of host-directed inhibitors of myxoviruses

2013 ◽  
Vol 9 ◽  
pp. 197-203 ◽  
Author(s):  
Terry W Moore ◽  
Kasinath Sana ◽  
Dan Yan ◽  
Pahk Thepchatri ◽  
John M Ndungu ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Asymmetric Synthesis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Aqueous Solubility ◽  
Influenza Viruses ◽  
Lead Compound ◽  
Synthetic Strategy ◽  
Aqueous Solubilities ◽  
Nanomolar Range

High-throughput screening (HTS) previously identified benzimidazole 1 (JMN3-003) as a compound with broad antiviral activity against different influenza viruses and paramyxovirus strains. In pursuit of a lead compound from this series for development, we sought to increase both the potency and the aqueous solubility of 1. Lead optimization has achieved compounds with potent antiviral activity against a panel of myxovirus family members (EC50 values in the low nanomolar range) and much improved aqueous solubilities relative to that of 1. Additionally, we have devised a robust synthetic strategy for preparing 1 and congeners in an enantio-enriched fashion, which has allowed us to demonstrate that the (S)-enantiomers are generally 7- to 110-fold more potent than the corresponding (R)-isomers.

scholarly journals A Novel Method—Amenable for High-throughput Screening Purposes—to Quantify Antiviral Activity Against Viruses that Induce Limited CPE

2011 ◽  
Vol 90 (2) ◽  
pp. A33-A34
Author(s):  
Dirk Jochmans ◽  
Bernadette G. van den Hoogen ◽  
Pieter Leyssen ◽  
Ron A. Fouchier ◽  
Johan Neyts
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Novel Method

scholarly journals Discovery of Novel Benzoquinazolinones and Thiazoloimidazoles, Inhibitors of Influenza H5N1 and H1N1 Viruses, from a Cell-Based High-Throughput Screen

2010 ◽  
Vol 16 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Joseph A. Maddry ◽  
Xi Chen ◽  
Colleen B. Jonsson ◽  
Subramaniam Ananthan ◽  
Judith Hobrath ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Active Compounds ◽  
Influenza Activity ◽  
A Cell ◽  
Molecular Libraries ◽  
Viral Cytopathic Effect

A highly reproducible and robust cell-based high-throughput screening (HTS) assay was adapted for screening of small molecules for antiviral activity against influenza virus strain A/Vietnam/1203/2004 (H5N1). The NIH Molecular Libraries Small Molecule Repository (MLSMR) Molecular Libraries Screening Centers Network (MLSCN) 100,000-compound library was screened at 50 µM. The “hit” rate (>25% inhibition of the viral cytopathic effect) from the single-dose screen was 0.32%. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen yielded 5 active compounds (SI value >3). One compound showed an SI50 value of greater than 3, 3 compounds had SI values ranging from greater than 14 to 34, and the most active compound displayed an SI value of 94. The active compounds represent 2 different classes of molecules, benzoquinazolinones and thiazoloimidazoles, which have not been previously identified as having antiviral/anti-influenza activity. These molecules were also effective against influenza A/California/04/2009 virus (H1N1) and other H1N1 and H5N1 virus strains in vitro but not H3N2 strains. Real-time qRT-PCR results reveal that these chemotypes significantly reduced M1 RNA levels as compared to the no-drug influenza-infected Madin Darby canine kidney cells.

scholarly journals Inducible APOBEC3G-Vif Double Stable Cell Line as a High-Throughput Screening Platform To Identify Antiviral Compounds

2009 ◽  
Vol 54 (1) ◽  
pp. 78-87 ◽  
Author(s):  
Boris Nowotny ◽  
Thomas Schneider ◽  
Gabriele Pradel ◽  
Tanja Schirmeister ◽  
Axel Rethwilm ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Yellow Fluorescent Protein ◽  
26S Proteasome ◽  
Stable Cell Line ◽  
Antiviral Compound ◽  
Compound Libraries ◽  
Antiviral Compounds

ABSTRACT Inhibition of the interaction of the human cytidine-deaminase APOBEC3G (A3G) with the human immunodeficiency virus (HIV) type 1-specific viral infectivity factor (Vif) represents a novel therapeutic approach in which a cellular factor with potent antiviral activity (A3G) plays a key role. In HIV-infected cells, the interaction of Vif with A3G leads to the subsequent degradation of A3G by the 26S proteasome via the ubiquitin pathway and to the loss of antiviral activity. To establish a stable and convenient cellular testing platform for the high-throughput screening of potential antiviral compound libraries, we engineered a double transgenic cell line constitutively expressing an enhanced yellow fluorescent protein expressor (EYFP-A3G) fusion as well as a Tet-Off controllable Vif protein. With this cell line, we were able to measure precisely the Vif-induced degradation of A3G in the presence of potential antiviral compounds in an easy-to-handle, robust, and practical high-throughput multiwell plate format with an excellent screening window coefficient (Z factor) of 0.67.

scholarly journals High Throughput Screening of FDA-Approved Drug Library Reveals the Compounds that Promote IRF3-Mediated Pro-Apoptotic Pathway Inhibit Virus Replication

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 442 ◽  
Author(s):  
Anna Glanz ◽  
Karan Chawla ◽  
Stephanie Fabry ◽  
Gayatri Subramanian ◽  
Julie Garcia ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transcriptional Activity ◽  
Viral Infections ◽  
Antiviral Effect ◽  
Apoptotic Pathway ◽  
Antiviral Genes ◽  
Wide Range ◽  
Infected Cells

Interferon (IFN) regulatory factor 3 (IRF3) is the key transcription factor for the induction of IFN and antiviral genes. The absence of antiviral genes in IRF3 deficiency leads to susceptibility to a wide range of viral infections. Previously, we uncovered a function for nontranscriptional IRF3 (nt-IRF3), RLR (RIG-I-like receptor)-induced IRF3-mediated pathway of apoptosis (RIPA), which triggers apoptotic killing of virus-infected cells. Using knock-in mice expressing a transcriptionally inactive, but RIPA-active, IRF3 mutant, we demonstrated the relative contribution of RIPA to host antiviral defense. Given that RIPA is a cellular antiviral pathway, we hypothesized that small molecules that promote RIPA in virus-infected cells would act as antiviral agents. To test this, we conducted a high throughput screen of a library of FDA-approved drugs to identify novel RIPA activators. Our screen identified doxorubicin as a potent RIPA-activating agent. In support of our hypothesis, doxorubicin inhibited the replication of vesicular stomatitis virus, a model rhabdovirus, and its antiviral activity depended on its ability to activate IRF3 in RIPA. Surprisingly, doxorubicin inhibited the transcriptional activity of IRF3. The antiviral activity of doxorubicin was expanded to flavivirus and herpesvirus that also activate IRF3. Mechanistically, doxorubicin promoted RIPA by activating the extracellular signal-regulated kinase (ERK) signaling pathway. Finally, we validated these results using another RIPA-activating compound, pyrvinium pamoate, which showed a similar antiviral effect without affecting the transcriptional activity of IRF3. Therefore, we demonstrate that the RIPA branch of IRF3 can be targeted therapeutically to prevent virus infection.

scholarly journals Pharmacologic profiling reveals lapatinib as a novel antiviral against SARS-CoV-2 in vitro

2020 ◽  
Author(s):  
M. H. Raymonda ◽  
J. H. Ciesla ◽  
M. Monaghan ◽  
J. Leach ◽  
G. Asantewaa ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
Treatment Options ◽  
Drug Repurposing ◽  
Preclinical Model ◽  
Human Tissues ◽  
Pulmonary Fibroblasts ◽  
Improve Treatment ◽  
Novel Treatment

SUMMARYThe emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but an effective antiviral therapy has yet to be discovered. To improve treatment options, we conducted a high-throughput drug repurposing screen to uncover compounds that block the viral activity of SARS-CoV-2. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed at 10 dose points (ranging from 20 μM to 1 nM). Our screening revealed several FDA-approved agents that act as novel antivirals that block both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 replication by over 50,000-fold without any toxicity and at doses readily achievable in human tissues. Further, both lapatinib and doramapimod could be combined with remdesivir to dramatically improve antiviral activity in cells. These findings reveal novel treatment options for people infected with SARS-CoV-2 that can be readily implemented during the pandemic.

scholarly journals Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening

2006 ◽  
Vol 51 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Weidong Hao ◽  
Koleen J. Herlihy ◽  
Noelle Jie Zhang ◽  
Shella A. Fuhrman ◽  
Chau Doan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Drug Discovery ◽  
Antiviral Activity ◽  
Cell Line ◽  
High Throughput ◽  
Firefly Luciferase ◽  
Peptide Cleavage ◽  
Reverse Transcription Pcr ◽  
Replicon Cell Line

ABSTRACT Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neor marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z′ value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 μM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals ≥50 and ≤40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

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