The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity.

Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “black box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

The extract of marine sponge Hyrtios communis was found to inhibit activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) in T47D human breast tumor cells. Bioassay-guided isolation led to the identification of six new (1–6) and five previously reported (7–11) sesterterpene analogues and two unrelated sesterterpenes. Two new sesterterpenes, thorectidaeolide A (1) and 4-acetoxythorectidaeolide A (2), and luffariellolide (11) were among the most potent inhibitors of hypoxia (1% O2)-induced HIF-1 activation (IC50 values of 3.2, 3.5, and 3.6 μM, respectively). Luffariellolide (11) exhibited a significant level of cytotoxicity that mirrored its HIF-1 inhibitory activity. Neither compound 1, compound 2, nor any of the other less active sesterterpenes suppressed breast tumor T47D or MDA-MB-231 cell viability.

The hypoxia-inducible factor-1 (HIF-1) transcription factor regulates cellular oxygen homeostasis. Agents that activate HIF-1 and downstream HIF targets represent potential drug leads for the prevention and/or treatment of ischemic disorders. In a search for small-molecule HIF-1 activators, 1936 marine invertebrate and algal extract samples (U.S. National Cancer Institute’s Open Repository) were evaluated for HIF-1 activation activity in a cell-based reporter assay. Bioassay-guided fractionation of two active extracts of the sponge Dactylospongia elegans afforded four new sesquiterpene quinones (2–5), one new sesquiterpene phenol (6), the known Golgi disruptor ilimaquinone (1), and three previously reported ilimaquinone analogues (7–9). While antiproliferative activity was observed at higher concentrations, the sesquiterpene quinones (1–3) possessing a 2-hydroxy-5-methoxy-1,4-benzoquinone moiety activated HIF-1 and increased the expression of HIF-1 target gene vascular endothelial growth factor (VEGF) in T47D cells.

Natural product drug discovery programs often rely on the use of silica (Si) gel, reversed-phase media, or size-exclusion resins (e.g., RP-C18, Sephadex LH-20) for compound purification. The synthetic polymer-based sorbent Diaion HP20SS (cross-linked polystyrene matrix) is used as an alternative to prepare purified natural product libraries. To evaluate the impact of chromatographic media on the isolation of biologically active, yet chromatographically unstable natural products, Diaion HP20SS was evaluated side-by-side with normal-phase sorbents for irreversible binding of extract constituents and their effects on bioactivity. An array of chemically diverse natural product-rich extracts was selected as a test panel, and a cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) was employed to monitor potential change(s) in bioactivity. Silica gel caused significant irreversible binding of three out of 10 extracts. Curcuma longa, Saururus cernuus, and Citrus reticulata extracts showed decreased HIF-1 inhibitory activity after elution through Si gel. An additional nonpolar column wash of HP20SS with EtOAc retained considerable bioactivities of active extracts. In general, Si gel produced the greatest loss of bioactivity. However, HP20SS elution reduced significantly HIF-1 inhibitory activity of certain extracts (e.g., Asimina triloba).

Tumor cells exhibit enhanced glucose consumption and lactate production even when supplied with adequate oxygen (a phenomenon known as the Warburg effect, or aerobic glycolysis). Pharmacological inhibition of aerobic glycolysis represents a potential tumor-selective approach that targets the metabolic differences between normal and malignant tissues. Human breast tumor MDA-MB-231 cells were used to develop an assay system to discover natural product-based glycolysis inhibitors. The assay employed was based on hypersensitivity to glycolytic inhibition in tumor cells treated with the mitochondrial electron transport inhibitor rotenone. Under such conditions, ATP supply, and hence cell viability, depends exclusively on glycolysis. This assay system was used to evaluate 10648 plant and marine organism extracts from the U.S. National Cancer Institute’s Open Repository. Bioassay-guided isolation of an active Moronobea coccinea extract yielded the new bis-geranylacylphloroglucinol derivative moronone (1). Compound 1 exhibited enhanced antiproliferative/cytotoxic activity in the presence of rotenone-imposed metabolic stress on tumor cells. Surprisingly, mechanistic studies revealed that 1 did not inhibit glycolysis, but rather functions as a protonophore that dissipates the mitochondrial proton gradient. In the presence of rotenone, tumor cells may be hypersensitive to protonophores due to increased ATP utilization by the ATP synthase.

The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1–7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1–7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC50 value of 0.2 μM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.

Over 20 000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 and 0.068 μM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods.

In an effort to identify natural product-based molecular-targeted antitumor agents, mammea-type coumarins from the tropical/subtropical plant Mammea americana were found to inhibit the activation of HIF-1 (hypoxia-inducible factor-1) in human breast and prostate tumor cells. In addition to the recently reported mammea E/BB (15), bioassay-guided fractionation of the active extract yielded 14 mammea-type coumarins including three new compounds, mammea F/BB (1), mammea F/BA (2), and mammea C/AA (3). The absolute configuration of C-1′ in 1 was determined by the modified Mosher’s method on a methylated derivative. These coumarins were evaluated for their effects on mitochondrial respiration, HIF-1 signaling, and tumor cell proliferation/viability. Acetylation of 1 afforded a triacetoxylated product (A-2) that inhibited HIF-1 activation with increased potency in both T47D (IC50 0.83 μM for hypoxia-induced) and PC-3 cells (IC50 0.94 μM for hypoxia-induced). Coumarins possessing a 6-prenyl-8-(3-methyloxobutyl) substituent pattern exhibited enhanced HIF-1 inhibitory effects. The O-methylated derivatives were less active at inhibiting HIF-1 and suppressing cell proliferation/viability. Mechanistic studies indicate that these compounds act as anionic protonophores that potently uncouple mitochondrial electron transport and disrupt hypoxic signaling.

The cytotoxic marine red algal metabolite thyrsiferol (1) was found to inhibit hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast tumor cells (66% inhibition at 3 μM). Compound 1 also suppressed hypoxic induction of HIF-1 target genes (VEGF, GLUT-1) at the mRNA level, and displayed tumor cell line-selective time-dependent inhibition of cell viability/proliferation. Mechanistic studies revealed that 1 selectively suppressed mitochondrial respiration at complex I (IC50 3 μM). Thyrsiferol represents a prototypical, structurally unique electron transport chain inhibitor. The apparent rotenone-like activity may contribute to the observed cytotoxicity of 1 and play an important role in Laurencia chemical defense.The cytotoxic Laurencia metabolite thyrsiferol (1) is a structurally unique mitochondrial complex I inhibitor (IC50 3 μM) that inhibits hypoxia-induced HIF-1 activation in T47D human breast tumor cells.

The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC50 values of 0.96 and 0.89 μM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 μM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlie their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines.

Products that contain twig extracts of pawpaw (Asimina triloba) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.02 μg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1α protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.

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 (IC50 1 nM) 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.

The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.

The lipid extract of the marine sponge Mycale sp. inhibited the activation of hypoxia-inducible factor-1 (HIF-1) in a human breast tumor T47D cell-based reporter assay. Bioassay-guided isolation and structure elucidation yielded 18 new lipophilic 2,5-disubstituted pyrroles and eight structurally related known compounds. The active compounds inhibited hypoxia-induced HIF activation with moderate potency (IC50 values <10 μM). Mechanistic studies revealed that the active compounds suppressed mitochondrial respiration by blocking NADH-ubiquinone oxidoreductase (complex I) at concentrations that inhibited HIF-1 activation. Under hypoxic conditions, reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. By inhibiting electron transport (or delivery) to complex III under hypoxic conditions, lipophilic Mycale pyrroles appear to disrupt mitochondrial ROS-regulated HIF-1 signaling.

A natural product chemistry-based approach was employed to discover small-molecule inhibitors of the important tumor-selective molecular target hypoxia-inducible factor-1 (HIF-1). Bioassay-guided isolation of an active lipid extract of a Saipan collection of the marine sponge Lendenfeldia sp. afforded the terpene-derived furanolipid furospongolide as the primary inhibitor of hypoxia-induced HIF-1 activation (IC50 2.9 μM, T47D breast tumor cells). The active component of the extract also contained one new cytotoxic scalarane sesterterpene and two previously reported scalaranes. Furospongolide blocked the induction of the downstream HIF-1 target secreted vascular endothelial growth factor (VEGF) and was shown to suppress HIF-1 activation by inhibiting the hypoxic induction of HIF-1α protein. Mechanistic studies indicate that furospongolide inhibits HIF-1 activity primarily by suppressing tumor cell respiration via the blockade of NADH-ubiquinone oxidoreductase (complex I)-mediated mitochondrial electron transfer.

The transcription factor hypoxia-inducible factor-1 (HIF-1) has emerged as a major antitumor molecular target. Inhibition of HIF-1 activation has been shown to suppress the growth, survival, and metastatic spread of hypoxic tumors. The NCI Open Repository of marine invertebrates and algae lipid extracts was evaluated for HIF-1 inhibitory activity in a T47D human breast tumor cell-based reporter assay. Bioassay-guided chromatographic separation of the active extract from the sponge Dendrilla nigra produced four new lamellarin-like phenolic pyrroles, which most closely resemble the structure of the known D. cactos compound lamellarin O. However, unlike lamellarins, the structures of neolamellarin A (1), neolamellarin B (2), 5-hydroxyneolamellarin B (3), and 7-hydroxyneolamellarin A (4) lack the carboxyl moiety at position C-2 of the substituted pyrrole ring and have a significantly different pattern of oxidation. Compound 4 was found to inhibit hypoxia-induced HIF-1 activation (IC50 1.9 µM) in T47D cells. Hypoxic induction of vascular endothelial growth factor (VEGF), a potent angiogenic factor and HIF-1 target gene, was also inhibited by 4 at the secreted protein level.

The lipid extract of an Indonesian Lendenfeldia sp. sponge inhibited hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D breast tumor cells. Chromatographic separation yielded the new substituted naphthalene dimer 1, the new furanolipid 2, and three known homoscalarane sesterterpenes, 3–5. Compounds 1 and 3–5 inhibited hypoxia-induced HIF-1 activation (IC50 values: 0.64–6.9 µM), but also reduced the viability of T47D and MDA-MB- 231 breast tumor cells. Compound 4 was the most potent and showed a unique tumor cell line selectivity in the NCI 60-cell line panel. The general cytotoxicity of these compounds precluded their further consideration as HIF-1 inhibitors.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that promotes tumor cell adaptation and survival under hypoxic conditions. HIF-1 is currently recognized as an important molecular target for anticancer drug discovery. The National Cancer Institute open repository of marine invertebrates and algae lipid extracts was evaluated using a T47D breast tumor cell-based reporter assay for HIF-1 inhibitory activity. Bioassay-guided fractionation of an active extract from a crinoid Comantheria rotula yielded seven benzo[g]chromen-4-one and benzo[h]chromen-4-one pigments (1–7). The structures of the new benzo[g]chromenone dimer 9,9′-oxybis-neocomantherin (1) and another new natural pigment 5 were deduced from spectroscopic and spectrometric data. The crinoid pigments significantly inhibited both hypoxia-induced and iron chelator-induced HIF-1 luciferase reporter activity in breast and prostate tumor cells. However, inhibition of HIF-1 in the reporter assay did not translate into a significant decrease in the expression of the downstream HIF-1 target, secreted vascular endothelial growth factor (VEGF). Compound 1 was found to inhibit tumor cell growth in the NCI 60-cell line panel (GI50 values of 1.6–18.2 µM), and compound 6 produced a unique pattern of tumor cell growth suppression. Five cell lines from different organs were hypersensitive to 6 (GI50 values of 0.29–0.62 µM), and three others were moderately sensitive (GI50 values of 2.2–5.1 µM), while the GI50 values for most other cell lines ranged from 20 to 47 µM. Crinoid benzo[g]chromenones were also found to scavenge radicals in a modified DPPH assay.