A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors

<div><p>Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein <i>in situ</i>. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.</p></div

Quantitation of protein synthesis and inhibition by cycloheximide.

<p>(A) ICW format: ACHN cells (3000 cells/well) were treated for 15 min ± cycloheximide followed by 30 min ± puromycin. GAPDH (green) and puromycin (red) signals were visualized as described in the text. (B) Puromycin and GAPDH signals from ICW (data from panel A, represented by circles) and western blot (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165192#pone.0165192.s002" target="_blank">S2 Fig</a>—represented by squares). Cells were pretreated with 0–100 μM cycloheximide (15 min) followed by puromycin (30 min) and quantitation. In a separate ICW experiment, cells were treated with cycloheximide for 4 h then puromycin (30 min) and quantitation (represented by triangles). Black symbols represent puromycin signal, open GAPDH. All values were normalized to vehicle controls. Error bars represent sd (n = 4).</p

Effects of protein synthesis inhibitors on protein synthesis, levels of cFLIP protein, and TRAIL- induced apoptosis.

<p>(A) ACHN cells were treated for 4 h with 1 μM of the indicated compound at which point cells were processed for ICW detection and quantitation of GAPDH (open bars), puromycin (light grey), or cFLIP (dark grey). In a parallel experiment, TRAIL was added after 4 h with compounds and cell survival assessed after 24 h by the XTT assay (black bars). Signals were normalized to control on the same plate (vehicle control = 100%). Error bars represent sd (n = 4 for puromycin and GAPDH; n = 3 for cFLIP; and 3 plates, duplicate wells per plate for compounds + TRAIL). (B) Caspase 8 activity was measured after 4 h treatment with 1 μM of the indicated compound followed by 4 h in the absence (open bars) or presence (black bars) of TRAIL and normalized (vehicle control = 1.0). Error bars represent sd (n = 3). compounds: ANS: anisomycin, CHX: cycloheximide, EME: emetine, GLA: glaucarubinone, VA: verrucarin A).</p

Simultaneous visualization of puromycylated proteins (red) and GAPDH (green).

<p>(A) western blot format: ACHN cells were pretreated for 15 min ± cycloheximide (100 μM final) followed by puromycin for the indicated times. After extraction, electrophoretic separation, and transfer (25 μg/lane), detection employed anti-puromycin and anti-GAPDH followed by near IR-fluorophore-labeled secondary antibodies. (B) ICW format: ACHN cells were plated in 384-well plates at the indicated densities and treated ± puromycin (30 min) followed by analysis using the indicated antibody concentrations and visualization.</p

Enigmazole Phosphomacrolides from the Marine Sponge Cinachyrella enigmatica

Enigmazole B (1) and four new analogues, cis-enigmazole B (2), dehydroenigmazole B (3), enigmimide B (4), and enigmimide A (5), were isolated from the marine sponge Cinachyrella enigmatica. Their planar structures were elucidated by detailed NMR and MS data analyses, which established 1–3 to be oxazole-substituted 18-membered phosphomacrolides, while 4 and 5 were oxazole ring-opened congeners. The relative and absolute configurations in 1 were determined by a combination of chemical transformations and spectroscopic analyses. Photooxidation of the oxazole moiety in 1 gave enigmimide B (4), thus establishing that 4 has the same absolute configuration of 1. Enigmazole B (1) along with analogues 2 and 3 showed cytotoxicity against murine IC-2 mast cells with IC50 values of 3.6–7.0 μM. The enigmimides (4 and 5) and dephosphoenigmazoles did not show cytotoxicity (IC50 > 10 μM), implying that both the oxazole moiety and the phosphate group are necessary for the cytotoxicity of the enigmazole class macrolides

Isobutylhydroxyamides from the Pericarp of Nepalese <i>Zanthoxylum armatum</i> Inhibit <i>NF1</i>-Defective Tumor Cell Line Growth

A neurofibromatosis type 1 (<i>NF1</i>)-based bioassay-guided phytochemical investigation on <i>Zanthoxylum armatum</i> collected in Nepal led to the isolation of new timuramides A–D (<b>1</b>–<b>4</b>) and six known sanshools (<b>5</b>–<b>10</b>). The structures of all compounds were established by using modern spectroscopic techniques, including 1D and 2D NMR analysis and comparison with previously reported data. Most of the compounds inhibited growth of an <i>Nf1</i>- and <i>p53</i>-deficient mouse glioma cell line at noncytotoxic concentrations

17β-Hydroxywithanolides as Sensitizers of Renal Carcinoma Cells to Tumor Necrosis Factor‑α Related Apoptosis Inducing Ligand (TRAIL) Mediated Apoptosis: Structure–Activity Relationships

Renal cell carcinoma (RCC) is a cancer with poor prognosis, and the 5-year survival rate of patients with metastatic RCC is 5–10%. Consequently, treatment of metastatic RCC represents an unmet clinical need. Screening of a 50 000-member library of natural and synthetic compounds for sensitizers of RCC cells to TRAIL-mediated apoptosis led to identification of the 17β-hydroxywithanolide (17-BHW), withanolide E (<b>1</b>), as a promising lead. To explore structure–activity relationships, we obtained natural and semisynthetic withanolides <b>1</b>, <b>2a</b>, <b>2c</b>, and <b>3</b>–<b>36</b> and compared their ability to sensitize TRAIL-mediated apoptosis in a panel of renal carcinoma cells. Our findings revealed that 17-BHWs with a α-oriented side chain are superior to known TRAIL-sensitizing withanolides belonging to withaferin A class with a β-oriented side chain and demonstrated that the 17-BHW scaffold can be modified to enhance sensitization of RCCs to TRAIL-mediated apoptosis, thereby assisting development of natural-product-inspired drugs to treat metastatic RCC

Synergistic TRAIL Sensitizers from <i>Barleria alluaudii</i> and <i>Diospyros maritima</i>

<i>Barleria alluaudii</i> and <i>Diospyros maritima</i> were both investigated as part of an ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, two naphthoquinone epoxides, 2,3-epoxy-2,3-dihydrolapachol (<b>1</b>) and 2,3-epoxy-2,3-dihydro-8-hydroxylapachol (<b>2</b>), both not previously isolated from natural sources, and the known 2-methylanthraquinone (<b>3</b>) were identified from <i>B. alluaudii.</i> Time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra were utilized to establish the absolute configuration of <b>1</b> and <b>2</b>. Additionally, five known naphthoquinone derivatives, maritinone (<b>4</b>), elliptinone (<b>5</b>), plumbagin (<b>6</b>), (+)-<i>cis</i>-isoshinanolone (<b>7</b>), and ethylidene-6,6′-biplumbagin (<b>8</b>), were isolated from <i>D. maritima</i>. Compounds <b>1</b>, <b>2</b>, and <b>4</b>–<b>6</b> showed varying levels of synergy with TRAIL. Maritinone (<b>4</b>) and elliptinone (<b>5</b>) showed the highest synergistic effect, with more than a 3-fold increase in activity observed with TRAIL than with compound alone

Flabelliferins A and B, Sesterterpenoids from the South Pacific Sponge <i>Carteriospongia flabellifera</i>

Two new sesterterpenoids named flabelliferins A (<b>1</b>) and B (<b>2</b>) were isolated from the lipophilic extract of the sponge <i>Cateriospongia flabellifera,</i> collected in the South Pacific near Vanuatu. The structure and absolute configuration of these two compounds were assigned by a combination of one- and two-dimensional NMR spectroscopy and by Mosher’s ester analysis. Flabelliferin A (<b>1</b>) has a rare 25-homocheilanthane carbon skeleton, while flabelliferin B (<b>2</b>) is a 24-nor-25-homoscalarane sesterterpenoid

Deguelins, Natural Product Modulators of NF1-Defective Astrocytoma Cell Growth Identified by High-Throughput Screening of Partially Purified Natural Product Extracts

A high-throughput screening assay for modulators of Trp53/NF1 mutant astrocytoma cell growth was adapted for use with natural product extracts and applied to a novel collection of prefractionated/partially purified extracts. Screening 68 427 samples identified active fractions from 95 unique extracts, including the terrestrial plant <i>Millettia ichthyotona</i>. Only three of these extracts showed activity in the crude extract form, thus demonstrating the utility of a partial purification approach for natural product screening. The NF1 screening assay was used to guide purification of active compounds from the <i>M. ichthyotona</i> extract, which yielded the two rotenones deguelin (<b>1</b>) and dehydrodeguelin (<b>2</b>). The deguelins have been reported to affect growth of a number of cancer cell lines. They potently inhibited growth of only one of a panel of NF1/Trp53 mutant murine astrocytoma cell lines, possibly related to epigenetic factors, but had no effect on the growth of normal astrocytes. These results suggest the potential utility of deguelins as tools for further investigating NF1 astrocytoma cell growth. These bioprobes were identified only as a result of screening partially purified natural product extracts