Novel Lobophorins Inhibit Oral Cancer Cell Growth and Induce <i>Atf4</i>- and <i>Chop</i>-Dependent Cell Death in Murine Fibroblasts

As part of the International Cooperative Biodiversity Groups (ICBG) Program, we were interested in identifying biologically active unfolded protein response (UPR) inducing compounds from marine microorganisms isolated from Costa Rican biota. With this aim in mind we have now generated more than 33,000 unique prefractionated natural product extracts from marine and terrestrial organisms that have been submitted to the Center of Chemical Genomics (CCG) at the University of Michigan for high throughput screening (HTS). An effective complementary cell-based assay to identify novel modulators of UPR signaling was used for screening extracts. Active fractions were iteratively subjected to reverse-phase HPLC chromatographic analysis, and together with lobophorin A, B, E, and F (1–4), three new lobophorin congeners, designated as CR1 (5), CR2 (6), and CR3 (7) were isolated. Herein, we report that secondary assays revealed that the new lobophorins induced UPR-associated gene expression, inhibited oral squamous cell carcinoma cell growth, and led to UPR-dependent cell death in murine embryonic fibroblast (MEF) cells

Antimycin A improves clinical disease and survival and reduces CNS titers in mice infected with WEEV.

<div><p>(A and B) Clinical disease severity and survival in WEEV-infected mice. C57BL/6 mice were infected with 10³ pfu WEEV, treated twice daily with DMSO or the indicated dose of antimycin A via intraperitoneal injection, and both clinical disease (A) and mortality (B) were monitored for 14 days post-infection. Representative results from one of two independent experiments are shown (N = 7-8 mice per group). *<i>p</i>-value < 0.05 compared to DMSO-treated mice.</p> <p>(C) Virus titers in the CNS of WEEV-infected mice. Mice were infected and treated as described above, and virus titers in brain were determined at 6 days post-infection. N = 4 mice per group. **p-value < 0.01 compared to DMSO control.</p></div

Schematic of marine microbe-based natural product extract production, screening, and validation.

<p>Individual steps are indicated in the left column, with explanatory comments provided on the right. The number of extracts and corresponding number of individual strains, where appropriate, are indicated in bold type between steps.</p

Antimycin A derivatives produced by <i>Streptomyces</i> have potent antiviral activity against WEEV serogroup alphaviruses.

<div><p>(A) Molecular structure of antimycin A. Core structure is shown at the top, and the individual R1 and R2 constituents of derivatives A1a, A2a, A3a, A4a, and A10a are shown below the core structure. Specific atom designations correspond to the NMR results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone.0082318.s005" target="_blank">Table S1</a>.</p> <p>(B) Antiviral activity of commercial antimycin A (AA) and mycophenolic acid (MPA) analyzed with WEEV replicons. Dose titration results for both replicon activity (closed symbols) and viability (open symbols) are presented as the percent untreated control cells and represent the mean ± SEM from at least five independent experiments. Calculated IC₅₀ values for anti-replicon activity are shown on the graph for both compounds, and an average MW of 550 g/mol was used to estimate molar concentrations for commercial antimycin A.</p> <p>(C and D) Antiviral activity of commercial AA and MPA analyzed with infectious WEEV (C) or FMV (D) in BE(2)-C neuronal cells. Cells were infected with WEEV (MOI = 0.1) or FMV (MOI = 1), treated simultaneously with compounds at the indicated concentrations, and virus production was measured by plaque assay at 24 hpi. Results are presented as infectious virion concentration in tissue culture supernatants and represent the mean ± SEM from at least three independent experiments. Calculated IC₅₀ values are shown on the graph for both compounds, and for commercial antimycin A these values were determined as described above in (B). The dashed reference lines represent results from infected cells treated with DMSO control.</p> <p>(E) HPLC separation of individual antimycin A derivatives from commercial stock compound. Only the select portion of an HPLC tracing that contained the four most prominent peaks is shown, and the various grey scale tracings represent different absorbance wavelengths. The identification of individual antimycin A derivatives represented by the four most prominent peaks is shown, where structures were determined by NMR analysis of purified fractions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone.0082318.s005" target="_blank">Table S1</a>).</p> <p>(F) Antiviral activity of individual antimycin A derivatives analyzed with WEEV replicons. Dose titration results are presented as the percent untreated control cells and represent the mean ± SEM from at least four independent experiments. Calculated IC₅₀ values for individual derivatives are shown on the graph, and were calculated using MWs of 548.63, 534.61, 520.58, and 506.55 g/mol for antimycins A1a, A2a, A3a, and A4a, respectively. The methoxy group in 2-methoxyantimycin A3 (MeO-AA3) is located at the 6’ position in the core antimycin structure shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082318#pone-0082318-g005" target="_blank">Figure 5A</a>. ND, not determined.</p></div

Disruption of mitochondrial electron transport suppresses WEEV replication.

<div><p>(A) Schematic of mETC enzyme complexes. The known targets for the inhibitors shown in italics are indicated by the cross bars. Cyt c, cytochrome C; CoQ, coenzyme Q.</p> <p>(B) Antiviral activity and toxicity of mETC inhibitors. Cells were treated with increasing concentrations of the indicated inhibitors, and replicon inhibition, total cellular ATP production, and cytotoxicity were measured in separate assays. Results are presented as IC₅₀ or CC₅₀ values for the indicated parameter, and represent the mean ± SEM from at least three independent experiments. The numerical values on the graph indicate fold-differences in IC₅₀ values between replicon inhibition and ATP production suppression for the indicated select compounds. For rotenone, the comparison was made with CC₅₀ values, since we were unable to calculate reliable IC₅₀ values for ATP production suppression.</p> <p>(C) Complementation assays with select mETC inhibitors and WEEV replicons. Cells were treated with 100 μM of the indicated supplement or antioxidant and antimycin A (AA), CCCP, or mycophenolic acid (MPA) at 2X or 5X replicon IC₅₀ concentrations, and replicon activity was measured 16-20 h later. Results represent the mean ± SEM from four independent experiments. <i>p</i>-value < 0.05* or 0.005** compared to supplement- or antioxidant-only treated controls. 2-MPG, <i>N</i>-(2-mercaptopropionyl)glycine.</p> <p>(D) Complementation assay with antimycin A and infectious virus. BE(2)-C cells were infected with FMV at an MOI = 1, treated simultaneously with 100 μM of the indicated supplement or antioxidant and control DMSO or antimycin A at 5X replicon IC₅₀ concentration, and viral titers in tissue culture supernatants were measured at 24 hpi. Results represent the mean ± SEM from four independent experiments. **<i>p</i>-value < 0.005 compared to inhibitor-treated controls without supplementation (open bars).</p></div

Purified antiviral compound from <i>S. kaviengensis</i> suppresses WEEV RNA replication and virus production in single-step growth assays.

<div><p>(A) Infectious virion production. BE(2)-C cells were infected with WEEV at an MOI = 10, treated with DMSO, 25 μM mycophenolic acid (MPA), or 100 ng/ml (~200 nM) purified compound F7E2e, and virus titers is tissue culture supernatants were determined by plaque assay at 6, 12, 24, and 48 hpi. Plaque assay sensitivity was 10² pfu/ml. Results represent the mean ± SEM from three independent experiments. *<i>p</i>-value < 0.05 compared to DMSO-treated controls for both MPA- and F7E2e-treated samples.</p> <p>(B) Quantitative RT-PCR analysis of WEEV RNA accumulation. Cells were infected and treated as above in (A), total RNA was harvested at the indicated time points, and primers corresponding to either the nsP1 or E1 WEEV genome were used to amplify and quantify either genomic (nsP1) or genomic plus subgenomic (E1) RNA accumulation. Results are presented as WEEV RNA levels relative to infected DMSO-treated control cells, and represent the mean ± SEM from six independent experiments. <i>p</i>-value < 0.001* or 0.0001** compared to DMSO-treated controls.</p> <p>(C) Northern blot analysis of WEEV RNA accumulation. Mock-infected cells (lane 1) or cells infected and treated as above in (A) with DMSO (lane 2), MPA (lane 3) or F7E2e (lane 4) were harvested at 12 hpi, and total RNA was analyzed by Northern blotting with a strand-specific ³²P-labelled riboprobe that detected both positive-sense genomic and subgenomic viral RNA (vRNA). The location and relative size of genomic and subgenomic vRNA are shown on the right, and the ethidium bromide-stained 28S rRNA band is shown as a loading control. Representative results from one of three independent experiments are shown.</p></div

Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and CH Functionalization

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon–carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%