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Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus.

Beran RK, Sharma R, Corsa AC, Tian Y, Golde J, Lundgaard G, Delaney WE, Zhong W, Greenstein AE - PLoS ONE (2012)

Bottom Line: By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth.In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation.Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Gilead Sciences, Foster City, California, United States of America.

ABSTRACT
During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.

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Effect of 17-AAG and CsA on HCV replication and viability determined by intracellular esterase activity.Antiviral activity (measured using the Renilla luciferase encoded by the HCV replicon; gray) and cell viability (measuring through the cleavage of calcein-AM by intracellular esterases; black) were assessed as a function of dose in a three day assay for the following molecules: (A) the HCV polymerase inhibitor HCV-796, (B) the ribosomal inhibitor puromycin, (C) the cyclophilin inhibitor CsA, (D) the microtubule inhibitor colchicine, (E) the anti-metabolite gemcitabine, and (F) the HSP90 inhibitor 17-AAG.
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pone-0030286-g001: Effect of 17-AAG and CsA on HCV replication and viability determined by intracellular esterase activity.Antiviral activity (measured using the Renilla luciferase encoded by the HCV replicon; gray) and cell viability (measuring through the cleavage of calcein-AM by intracellular esterases; black) were assessed as a function of dose in a three day assay for the following molecules: (A) the HCV polymerase inhibitor HCV-796, (B) the ribosomal inhibitor puromycin, (C) the cyclophilin inhibitor CsA, (D) the microtubule inhibitor colchicine, (E) the anti-metabolite gemcitabine, and (F) the HSP90 inhibitor 17-AAG.

Mentions: Antiviral replicon assays yielded standard dose-response curves ranging from 0 to 100% inhibition with EC50 values between 9 and 290 nM for all compounds. In contrast, the intracellular esterase activity assays yielded either no dose response (no toxicity), a full dose response (0 to 100% cell viability), or a partial dose response (e.g. cell viability was reduced, but reached a plateau between 0 and 50%). The HCV NS5B inhibitor HCV-796, a potent direct-acting antiviral [20], showed full antiviral activity without any observable cytotoxicity (Fig. 1A). The ribosome inhibitor puromycin (Fig. 1B), a known toxin [21], reduced intracellular esterase activity from 100% to 0% of the untreated control and fit a sigmoidal dose-response relationship. There was little difference between the apparent antiviral activity and cytotoxicity of puromycin in this assay (EC50 within 4-fold of CC50). The Cyclophilin inhibitor CsA [22] did exhibit cellular toxicity, but the concentrations required for the toxic effect were much higher (>20-fold) than the concentrations required for the antiviral effect (Fig. 1C). The microtubule inhibitor colchicine [23] (Fig. 1D) and the anti-metabolite gemcitabine [19](Fig. 1E), known cytostatic molecules, each demonstrated a full antiviral response with EC50 values of 9 and 12 nM, respectively (Table 1). However these compounds only partially reduced intracellular esterase activity at the majority of concentrations tested. The HSP90 inhibitor 17-AAG (Fig. 1F) demonstrated a full antiviral dose response with an EC50 of 12 nM (Table 1), but it had a complex, and biphasic effect on intracellular esterase activity. A partial reduction in intracellular esterase activity, similar to the colchicine and gemcitabine was observed at concentrations between 20 and 5,000 nM (Fig. 1D and E). While a CC50 value could be calculated in all cases (Table 1), the non-standard dose-response relationships observed may obscure the interpretation of these values. Colchicine, gemcitabine, and 17-AAG all interrupted viral replication with EC50 values between 9–12 nM (Table 1), but it appeared that all three compounds were also altering cellular viability at concentrations between 20 and 5,000 nM.


Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus.

Beran RK, Sharma R, Corsa AC, Tian Y, Golde J, Lundgaard G, Delaney WE, Zhong W, Greenstein AE - PLoS ONE (2012)

Effect of 17-AAG and CsA on HCV replication and viability determined by intracellular esterase activity.Antiviral activity (measured using the Renilla luciferase encoded by the HCV replicon; gray) and cell viability (measuring through the cleavage of calcein-AM by intracellular esterases; black) were assessed as a function of dose in a three day assay for the following molecules: (A) the HCV polymerase inhibitor HCV-796, (B) the ribosomal inhibitor puromycin, (C) the cyclophilin inhibitor CsA, (D) the microtubule inhibitor colchicine, (E) the anti-metabolite gemcitabine, and (F) the HSP90 inhibitor 17-AAG.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275588&req=5

pone-0030286-g001: Effect of 17-AAG and CsA on HCV replication and viability determined by intracellular esterase activity.Antiviral activity (measured using the Renilla luciferase encoded by the HCV replicon; gray) and cell viability (measuring through the cleavage of calcein-AM by intracellular esterases; black) were assessed as a function of dose in a three day assay for the following molecules: (A) the HCV polymerase inhibitor HCV-796, (B) the ribosomal inhibitor puromycin, (C) the cyclophilin inhibitor CsA, (D) the microtubule inhibitor colchicine, (E) the anti-metabolite gemcitabine, and (F) the HSP90 inhibitor 17-AAG.
Mentions: Antiviral replicon assays yielded standard dose-response curves ranging from 0 to 100% inhibition with EC50 values between 9 and 290 nM for all compounds. In contrast, the intracellular esterase activity assays yielded either no dose response (no toxicity), a full dose response (0 to 100% cell viability), or a partial dose response (e.g. cell viability was reduced, but reached a plateau between 0 and 50%). The HCV NS5B inhibitor HCV-796, a potent direct-acting antiviral [20], showed full antiviral activity without any observable cytotoxicity (Fig. 1A). The ribosome inhibitor puromycin (Fig. 1B), a known toxin [21], reduced intracellular esterase activity from 100% to 0% of the untreated control and fit a sigmoidal dose-response relationship. There was little difference between the apparent antiviral activity and cytotoxicity of puromycin in this assay (EC50 within 4-fold of CC50). The Cyclophilin inhibitor CsA [22] did exhibit cellular toxicity, but the concentrations required for the toxic effect were much higher (>20-fold) than the concentrations required for the antiviral effect (Fig. 1C). The microtubule inhibitor colchicine [23] (Fig. 1D) and the anti-metabolite gemcitabine [19](Fig. 1E), known cytostatic molecules, each demonstrated a full antiviral response with EC50 values of 9 and 12 nM, respectively (Table 1). However these compounds only partially reduced intracellular esterase activity at the majority of concentrations tested. The HSP90 inhibitor 17-AAG (Fig. 1F) demonstrated a full antiviral dose response with an EC50 of 12 nM (Table 1), but it had a complex, and biphasic effect on intracellular esterase activity. A partial reduction in intracellular esterase activity, similar to the colchicine and gemcitabine was observed at concentrations between 20 and 5,000 nM (Fig. 1D and E). While a CC50 value could be calculated in all cases (Table 1), the non-standard dose-response relationships observed may obscure the interpretation of these values. Colchicine, gemcitabine, and 17-AAG all interrupted viral replication with EC50 values between 9–12 nM (Table 1), but it appeared that all three compounds were also altering cellular viability at concentrations between 20 and 5,000 nM.

Bottom Line: By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth.In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation.Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Gilead Sciences, Foster City, California, United States of America.

ABSTRACT
During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth. In contrast, a cyclophilin inhibitor, cyclosporin A (CsA), exhibited selective antiviral activity without slowing cell proliferation. Furthermore, we observed that 17-AAG had little antiviral effect in a non-dividing cell-culture model of HCV replication, while CsA reduced HCV titer by more than two orders of magnitude in the same model. The assays we describe here are useful for discriminating selective antivirals from compounds that indirectly affect virus replication by reducing host cell viability or slowing cell growth.

Show MeSH
Related in: MedlinePlus