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 Early Clinical Evaluation of BMS-605339, a Potent and Orally Efficacious Tripeptidic Acylsulfonamide NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

The discovery of BMS-605339 (<b>35</b>), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropyl­acylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1′ site of the protease. The identification of <b>35</b> was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound <b>35</b> which demonstrated antiviral activity in HCV-infected patients