A truncated HIV Tat demonstrates potent and specific latency reversal activity.

A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.

Identification of Z-Tyr-Ala-CHN2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.

Development and optimization of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: Evaluation of 5676 Phase 1 Passed Structures.

Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.

An automated time-of-drug-addition assay to routinely determine the mode of action of HIV-1 inhibitors.

Cell-based high-throughput screening campaigns are widely used to identify novel antiviral compounds, for example, against human immunodeficiency virus type 1 (HIV-1). Typically, these assays enable identification of compounds that potentially target any viral or cellular factor involved in the viral replication cycle. Unraveling the mechanism of action of these active compounds is an important step to facilitate further drug development. Time-of-addition (TOA) assays are an elegant tool to achieve this goal by comparing the TOA profile of novel compounds with those of well-studied reference compounds. Downscaling to a 384-well format and automation significantly increase the capacity of the TOA assay, enabling compound handling around the clock. Mechanical liquid dispensing with optimized time points for compound addition ensures robustness (Z'>0.8) and maximal resolution in profiling novel antiviral compounds. The presented methodology has been optimized for routine use and allows for fully automated high-throughput screening to support the process in search for novel inhibitors of HIV-1.

A fluorescence-based high-throughput screening assay to identify HIV-1 inhibitors.

Highly active antiretroviral therapy (HAART) dramatically increases the long-term survival rates of human immunodeficiency virus type 1 (HIV-1) infected patients. Yet, poor adherence to therapy, adverse effects and the occurrence of resistant viruses can compromise the efficacy of HAART regiments. Therefore, there remains a clear unmet medical need for novel drugs and treatment options. In this chapter, we describe an HIV-1 antiviral high-throughput screening assay based on an HIV-1 permissive T lymphoblastoid MT4 cell line, stably transfected with a construct carrying an HIV-1 long terminal repeat promoter driving the expression of a reporter gene (enhanced green fluorescent protein). This assay runs in a 384-well format and enables the identification of HIV-1 inhibitors during a high-throughput screening campaign. In parallel, a cytotoxicity assay is performed to evaluate the compound-related in vitro toxicity.

A fluorescence-based high-throughput screening assay for identifying human cytomegalovirus inhibitors.

Human cytomegalovirus (HCMV) is a common opportunistic pathogen that can cause devastating -morbidity and mortality amongst neonates and immune-compromised patients. The current standard of care for HCMV infection is limited to four antiviral compounds that have major limitations in terms of long--term use, toxicity, and use during pregnancy. To provide patients with alternative treatment options to decrease HCMV-related morbidity and mortality, new drugs with novel modes of action are warranted. Here, we describe a validated high-throughput fluorescence antiviral screening assay based on infection of fibroblast cells with a fluorescently tagged reference strain of HCMV (AD169-GFP) to screen and profile HCMV inhibitors.

Antiviral activity and mode of action of TMC647078, a novel nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2'-deoxy-2'-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.

Development of robust antiviral assays for profiling compounds against a panel of positive-strand RNA viruses using ATP/luminescence readout.

The development of antiviral assays using an ATP/luminescence-based readout to profile antiviral compounds against the positive-strand RNA viruses: yellow fever virus (YFV), West Nile virus (WNV), Sindbis virus, and Coxsackie B virus, representing three virus families, is described. This assay readout is based upon the bioluminescent measurement of ATP in metabolically active cells. Antiviral efficacy was determined by measuring the ATP level in cells that were protected from the viral cytopathic effect (CPE) by the presence of antiviral compounds. The antiviral assay parameters were optimized and the assays were validated using a panel of different reference compounds to determine the intra- and inter-assay reproducibility. The signal-to-noise ratios for the yellow fever virus and West Nile virus assays were 7.5 and 36, respectively, comparing favorably with a signal-to-noise ratio of only 1.5 in the yellow fever virus neutral red dye uptake assay, an alternative readout for CPE inhibition. For Coxsackie B and Sindbis viruses, the signal-to-noise ratios were 40 and 50, respectively. These assays are robust, high-throughput, reproducible, and give much improved signal-to-noise ratios than those of dye uptake assays.

Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome-associated coronavirus.

The severity and global spread of the 2003 outbreak of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlighted the risks to human health posed by emerging viral diseases and emphasized the need for specific therapeutic agents instead of relying on existing broadly active antiviral compounds. The development of rapid screening assays is essential for antiviral drug discovery. Thus, a screening system for anti-SARS-CoV agents was developed, which evaluated compound potency, specificity and cytotoxicity at the initial screening phase. Cell lines were engineered to constitutively express an enhanced green fluorescent protein (EGFP) and used to detect (1) antiviral potency in SARS-CoV infection tests; (2) antiviral specificity in tests using the porcine coronavirus transmissible gastroenteritis virus (TGEV); and (3) cytotoxicity in the same assays without virus challenge. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. The suitability of this assay system in drug discovery was demonstrated by screening of 3388 small molecule compounds. The results show that these assays can be applied to high-throughput screening for identification of inhibitors selectively active against SARS-CoV.