The marine sponge metabolite mycothiazole: a novel prototype mitochondrial complex I inhibitor.

A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell line-dependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC(50) 1nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP(+), annonaceous acetogenins, piericidin A, and other complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound with a central thiazole moiety. The exquisite potency and structural novelty of 1 suggest that it may serve as a valuable molecular probe for mitochondrial biology and HIF-mediated hypoxic signaling

Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform

Kalkitoxin inhibits angiogenesis, disrupts cellular hypoxic signaling, and blocks mitochondrial electron transport in tumor cells

The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells

Recommended acquisition parameters.

<p>*Promotes hands-free centering of subjects.</p><p>**Filling the well to eliminate the meniscus effect (∼340 µL) produced the best results, however, this practice proved impractical for HTS.</p>+<p>Promotes maximal throughput and consistently high Z'-factors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029916#pone-0029916-t002" target="_blank">Table 2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029916#pone-0029916-t003" target="_blank">3</a>).</p>#<p>Lethality issues were evident at the 400 hertz level when volume was ≤100 µL.</p

Scan pattern Z'factors.

<p>*Lines were evaluated at 3 dpf or 6 dpf – i.e., once transgene expression was robust – with comparisons between global and regional lines per each reporter “color”; <i>q01</i> and <i>q02</i>, CFP, <i>mi2001</i> and <i>s356t</i>, GFP, <i>gmc701</i> and <i>q16a; q16b</i>, YFP, <i>vu234</i> and <i>q16a; c264</i>, RFP. n = 24 to 48 fish per assay.</p><p>**Comparison between subjects that were oriented by hand for optimal positioning to those that were dispensed in an automated fashion.</p

ARQiv detection of modulations in Notch pathway signaling.

<p>A) Comparison of fluorescence levels in the Notch-signaling transgenic reporter line, <i>Tp1:GFP</i> treated with increasing concentrations of DAPT (an inhibitor of gamma-secretase proteolytic activity). Following pre-treatment scans at 48 hpf, DAPT treatments commenced and post-treatment scans were taken every 24 hrs thereafter until 120 hpf. The results show a concomitant decrease in Notch signaling with increasing DAPT concentration. Paired t-tests showed that all DAPT treatments of 5 µM and above are significantly different (p-value ≤0.05) than controls from post-24 hr onward. B) Comparison of fluorescence levels in <i>Tp1:Cherry</i> embryos within the context of the Notch pathway mutant <i>mindbomb (mib).</i> The <i>mib</i> mutation is believed to abrogate Notch-signaling by preventing normal function of the ligand Delta. Embryos from a <i>mib</i>/+; <i>Tp1:Cherry</i> incross were phenotypically screened at 24hpf to delineate <i>mib</i> (-/-), from wildtype (wt, +/+) and heterozygotes (het, +/-), as well as to identify non-transgenic siblings. Reporter expression levels were then quantified every 12 hrs thereafter until 60 hpf. The wt transgenic embryos show a steady increase in Notch reporter expression; however, the <i>mib</i> transgenic embryos show a constant low expression level near that of non-transgenic controls. Paired t-tests showed revealed the <i>mib</i> embryos produced significantly lower (p-value ≤0.05) reporter levels than wt and het siblings starting at 24 hpf. C) Comparison of fluorescence levels in <i>Tp1:Cherry</i> embryos within the context of Notch pathway activation following induction of NICD expression in <i>Tp1:Cherry</i>; <i>hsp:Gal4; UAS:NICD</i> triple transgenic larvae. Controls included both non-heat shocked <i>Tp1:Cherry</i>⁺ siblings and heat-shocked <i>Tp1:Cherry</i>⁺ siblings in which <i>hsp:Gal4</i> and/or <i>UAS:NICD</i> was not present. Prior to heat shock a pre-treatment scan was performed at 4 dpf. An initial heat shock was performed to induce expression of NICD and repeated 12 hrs later. Scans were performed every 6 hrs over the next 24 hrs. Larvae with a slope of mCherry expression two standard deviations above non-heat shock controls were considered positive for all three transgenes and pooled. Larvae below this cut-off were considered to be negative for at least one of the other transgenes and pooled as heat-shocked negative controls. The data show that over-expression of NICD leads to a significant increase in expression of the mCherry reporter at 24 hours (p-value = 8.3E⁻⁰⁶) post heat-shock in keeping with ligand-independent activation of the Notch pathway.</p