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Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

Ulferts R, de Boer SM, van der Linden L, Bauer L, Lyoo HR, Maté MJ, Lichière J, Canard B, Lelieveld D, Omta W, Egan D, Coutard B, van Kuppeveld FJ - Antimicrob. Agents Chemother. (2016)

Bottom Line: All compounds acted through the inhibition of genome replication.Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds.Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases and Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Screening controls and overall screening data. (A) Data showing levels of infection based on intensity of GFP staining. Shown are box-and-whisker plots for screening controls and samples. All data were normalized against the negative controls in each plate. The lot displays the median, interquartile ranges (IQR), and outliers (outside 1.5 times the interquartile range). (B) Scatter plot showing data for all individual samples and controls. The regression line was plotted based on a least-squares criterion.
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Figure 1: Screening controls and overall screening data. (A) Data showing levels of infection based on intensity of GFP staining. Shown are box-and-whisker plots for screening controls and samples. All data were normalized against the negative controls in each plate. The lot displays the median, interquartile ranges (IQR), and outliers (outside 1.5 times the interquartile range). (B) Scatter plot showing data for all individual samples and controls. The regression line was plotted based on a least-squares criterion.

Mentions: To identify compounds with antiviral activity against enteroviruses, we screened the Prestwick Chemical Library, a library consisting of 1,200 approved drugs (U.S. Food and Drug Administration, European Medicines Agency, or other agency), for compounds that inhibit the replication of CV-B3 by using the CV-B3–GFP reporter virus. This reporter virus encodes GFP upstream of the capsid coding region. The intracellular GFP expression levels in CV-B3–GFP-infected cells thus correlate with virus replication (13). Cells were pretreated with 10 μM compound and infected with virus, and GFP expression was analyzed at 6 h p.i. This setup allows the detection of compounds that affect entry and replication but not of those that affect assembly. The known enterovirus inhibitors ribavirin (20 μM and 60 μM) and guanidine hydrochloride (GuHCl) (2 mM) were included as positive controls. The screen was performed twice with three replicates of each library compound. Ribavirin and GuHCl strongly decreased the mean average enhanced GFP (EGFP) fluorescence intensity, consistent with their known antiviral activity (Fig. 1A and B). Compounds that reduced the median average fluorescence intensity >20-fold and resulted in <10% reduction of the median number of DAPI-positive cells (as a measure of cell viability) in both screens were considered primary hits. These compounds were pirlindole mesylate, fluoxetine hydrochloride, formoterol fumarate, dibucaine, and zuclopenthixol hydrochloride (Table 1). The identified drugs are of diverse structures, belonging to diverse chemical classes, and are known to target different cellular factors. Formoterol is an agonist of β2 adrenergic receptors (β2-ARs) that is currently used as a bronchodilator for the treatment of asthma. Dibucaine is in use as a topical anesthetic that acts through the inhibition of voltage-activated sodium channels. Pirlindole is an inhibitor of monoamine oxidase A (MAO-A) (21), an enzyme involved in the metabolism of monoamines, including serotonin, melatonin, adrenaline, and noradrenaline. Zuclopenthixol is an antagonist of D2 dopamine receptors and used for the treatment of psychotic disorders. Fluoxetine HCl (Prozac) is a selective serotonin reuptake inhibitor used to treat depression. We and others previously identified fluoxetine in independent screens as an inhibitor of EV-B and EV-D members, and we showed that the compound acts on viral protein 2C (9, 10). Thus, these four compounds, formoterol, pirlindole mesylate, dibucaine, and zuclopenthixol, were analyzed further.


Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

Ulferts R, de Boer SM, van der Linden L, Bauer L, Lyoo HR, Maté MJ, Lichière J, Canard B, Lelieveld D, Omta W, Egan D, Coutard B, van Kuppeveld FJ - Antimicrob. Agents Chemother. (2016)

Screening controls and overall screening data. (A) Data showing levels of infection based on intensity of GFP staining. Shown are box-and-whisker plots for screening controls and samples. All data were normalized against the negative controls in each plate. The lot displays the median, interquartile ranges (IQR), and outliers (outside 1.5 times the interquartile range). (B) Scatter plot showing data for all individual samples and controls. The regression line was plotted based on a least-squares criterion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4862474&req=5

Figure 1: Screening controls and overall screening data. (A) Data showing levels of infection based on intensity of GFP staining. Shown are box-and-whisker plots for screening controls and samples. All data were normalized against the negative controls in each plate. The lot displays the median, interquartile ranges (IQR), and outliers (outside 1.5 times the interquartile range). (B) Scatter plot showing data for all individual samples and controls. The regression line was plotted based on a least-squares criterion.
Mentions: To identify compounds with antiviral activity against enteroviruses, we screened the Prestwick Chemical Library, a library consisting of 1,200 approved drugs (U.S. Food and Drug Administration, European Medicines Agency, or other agency), for compounds that inhibit the replication of CV-B3 by using the CV-B3–GFP reporter virus. This reporter virus encodes GFP upstream of the capsid coding region. The intracellular GFP expression levels in CV-B3–GFP-infected cells thus correlate with virus replication (13). Cells were pretreated with 10 μM compound and infected with virus, and GFP expression was analyzed at 6 h p.i. This setup allows the detection of compounds that affect entry and replication but not of those that affect assembly. The known enterovirus inhibitors ribavirin (20 μM and 60 μM) and guanidine hydrochloride (GuHCl) (2 mM) were included as positive controls. The screen was performed twice with three replicates of each library compound. Ribavirin and GuHCl strongly decreased the mean average enhanced GFP (EGFP) fluorescence intensity, consistent with their known antiviral activity (Fig. 1A and B). Compounds that reduced the median average fluorescence intensity >20-fold and resulted in <10% reduction of the median number of DAPI-positive cells (as a measure of cell viability) in both screens were considered primary hits. These compounds were pirlindole mesylate, fluoxetine hydrochloride, formoterol fumarate, dibucaine, and zuclopenthixol hydrochloride (Table 1). The identified drugs are of diverse structures, belonging to diverse chemical classes, and are known to target different cellular factors. Formoterol is an agonist of β2 adrenergic receptors (β2-ARs) that is currently used as a bronchodilator for the treatment of asthma. Dibucaine is in use as a topical anesthetic that acts through the inhibition of voltage-activated sodium channels. Pirlindole is an inhibitor of monoamine oxidase A (MAO-A) (21), an enzyme involved in the metabolism of monoamines, including serotonin, melatonin, adrenaline, and noradrenaline. Zuclopenthixol is an antagonist of D2 dopamine receptors and used for the treatment of psychotic disorders. Fluoxetine HCl (Prozac) is a selective serotonin reuptake inhibitor used to treat depression. We and others previously identified fluoxetine in independent screens as an inhibitor of EV-B and EV-D members, and we showed that the compound acts on viral protein 2C (9, 10). Thus, these four compounds, formoterol, pirlindole mesylate, dibucaine, and zuclopenthixol, were analyzed further.

Bottom Line: All compounds acted through the inhibition of genome replication.Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds.Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases and Immunology, Virology Division, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus