A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses

<div><p>A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC₅₀) values of 2 to 8 μM relative to IC₅₀ values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.</p></div

Chemical structure of the quinoxaline Compound 1.

<p>The activity of Compound 1 was tested in dose-response assays against a broader panel of cell lines, including both HPV transformed cells (Ca Ski, SiHa, HeLa, SCHPV-18, SCHPV-31, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155909#pone.0155909.g002" target="_blank">Fig 2A</a>) and HPV-negative cells (C33a, HaCat, Saos-2, HCT-116, J2-3T3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155909#pone.0155909.g002" target="_blank">Fig 2B</a>).</p

Susceptibility of HPV-positive and -negative cell lines to Compound 1.

<p>Dose response curves for HPV transformed cell lines (A) or HPV-negative cell lines (B) treated with Compound 1. The y axes depict percent inhibition of cell proliferation in viability assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155909#sec002" target="_blank">Materials and Methods</a>). Graphs depict a typical experiment with duplicates. IC₅₀ values represent the average of ≥3 independently determined values, with standard deviation of these values in paretheses. (C) Colonies formed by the indicated HPV-positive or HPV-negative cell lines in the absence (DMSO) or presence of 3 or 12 μM Compound 1.</p

Effects of Compound 1 on activation of Caspases 8 and 9.

<p>(A) The diagram depicts the full-length Procaspase-8 and Procaspase-9 proteins and their cleaved products formed upon activation (adapted from[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155909#pone.0155909.ref035" target="_blank">35</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155909#pone.0155909.ref036" target="_blank">36</a>]). (B) Ca Ski cells were treated with DMSO, Compound 1 (24 or 64 μM), 0.1 μg/mL TRAIL or 5 μM camptothecin for the indicated times. Western blots of lysates from treated cells were probed with antibodies directed against the Caspase-8 cleavage products, p41 and p43 (top panel), Procaspase-9 and its cleaved forms, p35 and p37 (middle panel) or Actin (bottom panel) as a control for sample loading.</p

Effect of Compound 1 on markers of apoptosis in treated cells.

<p>(A) HPV-positive and HPV-negative cell lines were treated with increasing concentrations of Compound 1 for 6 hours, and then tested for Caspase-3/7 activity. (B) Ca Ski cells were treated with the indicated concentrations of Compound 1 and tested for Caspase-3/7 activity at various times. The graph shows mean values of percent Caspase-3/7 activity relative to DMSO-treated controls. Each point represents the mean of triplicates, with error bars indicating standard deviation. (C) Ca Ski cells were treated with DMSO or the indicated concentrations of Compound 1 for 6 or 24 hours, and then stained with Annexin-V and 7-AAD prior to flow cytometry. (D) HPV-positive Ca Ski or HPV-negative C33a cells treated for 24 hours with DMSO, 18 μM Compound 1, or the control inhibitors 5 μM nocodazole or 1 μM camptothecin. Cells were fixed and stained with either the DNA stain Hoechst or by TUNEL, as indicated.</p

Effect of Compound 1 on protein expression in treated cells.

<p>Lysates from Ca Ski cells treated with DMSO or 12, 18 and 24 μM Compound 1 (A) or 5, 10, 20 and 40 nM bortezomib (B) were separated by SDS-PAGE and probed with antibodies against the specific proteins listed, as described in Materials and Methods. Western blots of Compound 1 treated HPV-negative cell lines HCT-116 and HaCat are shown in panels C and D.</p

Discovery and Preclinical Characterization of the Cyclopropylindolobenzazepine BMS-791325, A Potent Allosteric Inhibitor of the Hepatitis C Virus NS5B Polymerase

Described herein are structure–activity relationship studies that resulted in the optimization of the activity of members of a class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors. Subsequent iterations of analogue design and syntheses successfully addressed off-target activities, most notably human pregnane X receptor (hPXR) transactivation, and led to significant improvements in the physicochemical properties of lead compounds. Those analogues exhibiting improved solubility and membrane permeability were shown to have notably enhanced pharmacokinetic profiles. Additionally, a series of alkyl bridged piperazine carboxamides was identified as being of particular interest, and from which the compound BMS-791325 (<b>2</b>) was found to have distinguishing antiviral, safety, and pharmacokinetic properties that resulted in its selection for clinical evaluation