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

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.

Figure 1. 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.

Figure 2. Effect of 17-AAG and CsA on cellular viability determined by measuring intracellular ATP concentration.

Cell viability (measured by determining intracellular ATP levels) was 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.

Figure 3. Effect of 17-AAG and CsA on cellular viability determined by cell count.

Cell viability (measured by direct microscopic quantification of Hoechst-stained nuclei) was 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. Colchicine dramatically altered cellular morphology, preventing an accurate cell count.

Figure 4. 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.

Figure 5. 17-AAG slows cellular growth at its effective antiviral concentration.

(A) Cellular growth was determined by measuring the percent confluence (by microscopic quantification) every 4 hours. HCV-796 (blue diamonds), CsA (pink squares), puromycin (black triangles), 17-AAG (green triangles). (B) Growth rate (% confluence per day) as a function of compound dose. HCV-796 (blue diamonds), CsA (pink squares), puromycin (black triangles), 17-AAG (green triangles). (C) Trypan-blue exclusion was used to determine the number of viable cells each day post drug addition. HCV-796 (blue diamonds), CsA (pink squares), puromycin (black triangles), 17-AAG (green triangles), or an equivalent volume of DMSO (grey circles).

Figure 6. 17-AAG reduces HCV titer by slowing cellular growth.

(A) Non-dividing HCV-infected cultures were treated with 10×EC₅₀ for each compound. Subsequently, extracellular HCV titer was quantified at various times in the presence of DMSO (circles), 0.35 µM HSP90 inhibitor 17-AAG (triangles), 1.30 µM cyclosporin inhibitor CsA (squares), or 1.40 µM HCV protease inhibitor BILN-2061 (diamonds). The DMSO data were fit to a linear equation. The CsA and BILN-2061 data were fit to second-order exponential equations. The 17-AAG data could not be fit to first or second order exponential equations. (B) Dividing HCV-infected cultures were treated with 10×EC₅₀ compound. Subsequently, extracellular HCV titer was quantified at various times in the presence of DMSO (circles), 0.35 µM HSP90 inhibitor 17-AAG (triangles), 1.30 µM cyclophilin inhibitor CsA (squares), or 1.40 µM HCV protease inhibitor BILN-2061 (diamonds). The DMSO data were fit to a linear equation. The CsA and BILN-2061 data were fit to second-order exponential equations. The 17-AAG data could not be fit to first or second order exponential equations.