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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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Visual assessment of replicon cell number and morphology after 1 µM compound treatment.Hoechst-stained nuclei were visualized after treatment for three days with 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-g004: Visual assessment of replicon cell number and morphology after 1 µM compound treatment.Hoechst-stained nuclei were visualized after treatment for three days with 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: To better understand the effects of each compound on cell number and morphology, images of cells treated with 1 µM compound were visually inspected (Fig. 4). 1 µM puromycin eliminated all cells in the course of the three-day assay (Fig. 4B). 1 µM 17-AAG (Fig. 4F) or gemcitabine (Fig. 4E) reduced the number of cells per image to ∼22% of the controls. Neither 1 µM CsA (Fig. 4C) nor HCV-796 (Fig. 4A) altered the number of cells per image when compared to the no-drug control (Fig. 4D). In agreement with the intracellular ATP assay data, these data suggested that the antiviral effect of puromycin, gemcitabine, and 17-AAG was a consequence of adverse effects on cell health or proliferation. In contrast, CsA and HCV-796 display antiviral activity at concentrations that do not alter cell number.


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)

Visual assessment of replicon cell number and morphology after 1 µM compound treatment.Hoechst-stained nuclei were visualized after treatment for three days with 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-g004: Visual assessment of replicon cell number and morphology after 1 µM compound treatment.Hoechst-stained nuclei were visualized after treatment for three days with 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: To better understand the effects of each compound on cell number and morphology, images of cells treated with 1 µM compound were visually inspected (Fig. 4). 1 µM puromycin eliminated all cells in the course of the three-day assay (Fig. 4B). 1 µM 17-AAG (Fig. 4F) or gemcitabine (Fig. 4E) reduced the number of cells per image to ∼22% of the controls. Neither 1 µM CsA (Fig. 4C) nor HCV-796 (Fig. 4A) altered the number of cells per image when compared to the no-drug control (Fig. 4D). In agreement with the intracellular ATP assay data, these data suggested that the antiviral effect of puromycin, gemcitabine, and 17-AAG was a consequence of adverse effects on cell health or proliferation. In contrast, CsA and HCV-796 display antiviral activity at concentrations that do not alter cell number.

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