Cellular Growth Kinetics Distinguish a Cyclophilin Inhibitor from an HSP90 Inhibitor as a Selective Inhibitor of Hepatitis C Virus

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

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

<p>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.</p

Effect of 17-AAG and CsA on HCV replication and viability determined by intracellular esterase activity.

<p>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.</p

Visual assessment of replicon cell number and morphology after 1 µM compound treatment.

<p>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.</p

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

<p>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.</p

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

<p>(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).</p

Substituted Indazoles as Na_v1.7 Blockers for the Treatment of Pain

The genetic validation for the role of the Na_v1.7 voltage-gated ion channel in pain signaling pathways makes it an appealing target for the potential development of new pain drugs. The utility of nonselective Na_v blockers is often limited due to adverse cardiovascular and CNS side effects. We sought more selective Na_v1.7 blockers with oral activity, improved selectivity, and good druglike properties. The work described herein focused on a series of 3- and 4-substituted indazoles. SAR studies of 3-substituted indazoles yielded analog <b>7</b> which demonstrated good in vitro and in vivo activity but poor rat pharmacokinetics. Optimization of 4-substituted indazoles yielded two compounds, <b>27</b> and <b>48</b>, that exhibited good in vitro and in vivo activity with improved rat pharmacokinetic profiles. Both <b>27</b> and <b>48</b> demonstrated robust activity in the acute rat monoiodoacetate-induced osteoarthritis model of pain, and subchronic dosing of <b>48</b> showed a shift to a lower EC₅₀ over 7 days

Substituted Indazoles as Na_v1.7 Blockers for the Treatment of Pain

The genetic validation for the role of the Na_v1.7 voltage-gated ion channel in pain signaling pathways makes it an appealing target for the potential development of new pain drugs. The utility of nonselective Na_v blockers is often limited due to adverse cardiovascular and CNS side effects. We sought more selective Na_v1.7 blockers with oral activity, improved selectivity, and good druglike properties. The work described herein focused on a series of 3- and 4-substituted indazoles. SAR studies of 3-substituted indazoles yielded analog <b>7</b> which demonstrated good in vitro and in vivo activity but poor rat pharmacokinetics. Optimization of 4-substituted indazoles yielded two compounds, <b>27</b> and <b>48</b>, that exhibited good in vitro and in vivo activity with improved rat pharmacokinetic profiles. Both <b>27</b> and <b>48</b> demonstrated robust activity in the acute rat monoiodoacetate-induced osteoarthritis model of pain, and subchronic dosing of <b>48</b> showed a shift to a lower EC₅₀ over 7 days