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1.  The Alternative Medicine Pawpaw and Its Acetogenin Constituents Suppress Tumor Angiogenesis via the HIF-1/VEGF Pathway 
Journal of natural products  2010;73(5):956-961.
Products that contain twig extracts of pawpaw (Asimina triloba, Annonaceae) 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.
doi:10.1021/np100228d
PMCID: PMC2890309  PMID: 20423107
2.  The Caulerpa Pigment Caulerpin Inhibits HIF-1 Activation and Mitochondrial Respiration 
Journal of natural products  2009;72(12):2104-2109.
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.
doi:10.1021/np9005794
PMCID: PMC2798910  PMID: 19921787
3.  Lipophilic 2,5-Disubstituted Pyrroles from the Marine Sponge Mycale sp. Inhibit Mitochondrial Respiration and HIF-1 Activation 
Journal of natural products  2009;72(11):1927-1936.
The lipid extract of the marine sponge Mycale sp. inhibited the activation of hypoxiainducible 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.
doi:10.1021/np900444m
PMCID: PMC2868385  PMID: 19845338
4.  Molecular-Targeted Antitumor Agents 15: Neolamellarins from the Marine Sponge Dendrilla nigra Inhibit Hypoxia-Inducible Factor-1 (HIF-1) Activation and Secreted Vascular Endothelial Growth Factor (VEGF) Production in Breast Tumor Cells 
Journal of natural products  2007;70(11):1741-1745.
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.
doi:10.1021/np070206e
PMCID: PMC2914556  PMID: 17958397
5.  Hypoxia-Selective Antitumor Agents: Norsesterterpene Peroxides from the Marine Sponge Diacarnus levii Preferentially Suppress the Growth of Tumor Cells under Hypoxic Conditions 
Journal of natural products  2007;70(1):130-133.
As part of an ongoing research program to discover natural products that suppress the hypoxia-activated tumor survival pathways, the lipid extract of the Papua New Guinea marine sponge Diacarnus levii was found to suppress hypoxia-induced HIF-1 activation and hypoxic tumor cell survival. Bioassay-guided isolation of D. levii yielded four new norsesterterpene peroxides, diacarnoxides A – D. Diacarnoxide B exhibits a significantly enhanced ability to suppress the growth of tumor cells under hypoxic conditions.
doi:10.1021/np0604883
PMCID: PMC2910712  PMID: 17253866
6.  Laurenditerpenol, a New Diterpene from the Tropical Marine Alga Laurencia intricata Potently Inhibits HIF-1 Mediated Hypoxic Signaling in Breast Tumor Cells 
Journal of natural products  2004;67(12):2002-2007.
The degree of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) promotes tumor cell adaptation and survival under hypoxic conditions. Therefore, specific HIF-1 inhibitors represent an important new class of potential tumor-selective therapeutic agents. A T47D human breast tumor cell-based reporter assay was used to examine extracts of plants and marine organisms for inhibitors of HIF-1 activation. Bioassay-guided fractionation of the lipid extract of the red alga Laurencia intricata yielded a structurally novel diterpene laurenditerpenol (1). The structure of 1 was determined spectroscopically. The relative configurations of the substituents of each ring system were assigned based on NOESY correlations. The absolute configurations of positions C-1 was determined by the modified Mosher ester procedure (directly in NMR tubes). Compound 1 potently inhibited hypoxia-activated HIF-1 (IC50: 0.4 μM) and hypoxia-induced VEGF (a potent angiogenic factor) in T47D cells. Compound 1 selectively inhibits HIF-1 activation by hypoxia but not iron chelator induced activation. Further, 1 suppresses tumor cell survival under hypoxic conditions without affecting normoxic cell growth. Compound 1 inhibits HIF-1 by blocking the induction of the oxygen-regulated HIF-1α protein. Mitochondrial respiration studies revealed that 1 suppresses oxygen consumption.
doi:10.1021/np049753f
PMCID: PMC2910713  PMID: 15620241
7.  Benzochromenones from the Marine Crinoid Comantheria rotula Inhibit Hypoxia-Inducible Factor-1 (HIF-1) in Cell-Based Reporter Assays and Differentially Suppress the Growth of Certain Tumor Cell Lines 
Journal of natural products  2007;70(9):1462-1466.
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 anti-cancer drug discovery. The NCI 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 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 1.6 to 18.2 μM) and 6 produced a unique pattern of tumor cell growth suppression. Five cell lines from different organs were hypersensitive to 6 (GI50 values 0.29 to 0.62 μM) and three others were moderately sensitive (GI50 values 2.2 to 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.
doi:10.1021/np070224w
PMCID: PMC2910718  PMID: 17844994
8.  Sodwanone and Yardenone Triterpenes from a South African Species of the Marine Sponge Axinella Inhibit Hypoxia-Inducible Factor-1 (HIF-1) Activation in both Breast and Prostate Tumor Cells 
Journal of natural products  2006;69(12):1715-1720.
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 anti-cancer drug discovery. A T47D breast tumor cell-based reporter assay was used to evaluate the NCI Open Repository of marine invertebrates and algae lipid extracts for HIF-1 inhibitory activity. Bioassay-guided fractionation and isolation of an active extract from Axinella sp. yielded seven new sodwanone triterpenoids [3-epi-sodwanone K (1), 3-epi-sodwanone K 3-acetate (2), 10,11-dihydrosodwanone B (4), sodwanones T–W (3, 7, 8, 9), the new yardenone triterpene 12R-hydroxyyardenone (10), and the previously reported compounds sodwanone A (5), sodwanone B (6), and yardenone (11). The structures and relative configurations of these Axinella metabolites were determined spectroscopically. The absolute configuration of 1 was determined by the modified Mosher ester procedure. Sodwanone V (8) inhibited both hypoxia-induced and iron chelator (1,10-phenanthroline)-induced HIF-1 activation in T47D breast tumor cells (IC50 15 μM) and 8 was the only sodwanone that inhibited HIF-1 activation in PC-3 prostate tumor cells (IC50 15 μM). Compounds 1, 3, 4, and 5 inhibited hypoxia-induced HIF-1 activation in T47D cells (IC50 values 20-25 μM). Compound 2 was cytotoxic to T47D cells (IC50 22 μM) and 8 showed cytotoxicity to MDA-MB-231 breast tumor cells (IC50 23 μM).
doi:10.1021/np060278q
PMCID: PMC2908379  PMID: 17190448
9.  The Terpenoid Tetrahydroisoquinoline Alkaloids Emetine, Klugine, and Isocephaeline Inhibit the Activation of Hypoxia-Inducible Factor-1 (HIF-1) in Breast Tumor Cells 
Journal of natural products  2005;68(6):947-950.
Klugine (1), isocephaeline (2), and emetine (4) inhibited hypoxia-inducible factor-1 (HIF-1) activation by hypoxia in T47D breast tumor cells (IC50 values 0.2, 1.1, and 0.11 µM, respectively). Compounds 1, 2, and 4 inhibited both hypoxia- and iron chelator-induced HIF-1 activation by blocking HIF-1α protein accumulation.
doi:10.1021/np050029m
PMCID: PMC2907142  PMID: 15974627
10.  Natural Product-Derived Small Molecule Activators of Hypoxia-Inducible Factor-1 (HIF-1) 
Current pharmaceutical design  2006;12(21):2673-2688.
Hypoxia-inducible factor-1 (HIF-1) is a key mediator of oxygen homeostasis that was first identified as a transcription factor that is induced and activated by decreased oxygen tension. Upon activation, HIF-1 upregulates the transcription of genes that promote adaptation and survival under hypoxic conditions. HIF-1 is a heterodimer composed of an oxygen-regulated subunit known as HIF-1α and a constitutively expressed HIF-1β subunit. In general, the availability and activity of the HIF-1α subunit determines the activity of HIF-1. Subsequent studies have revealed that HIF-1 is also activated by environmental and physiological stimuli that range from iron chelators to hormones. Preclinical studies suggest that HIF-1 activation may be a valuable therapeutic approach to treat tissue ischemia and other ischemia/hypoxia-related disorders.
The focus of this review is natural product-derived small molecule HIF-1 activators. Natural products, relatively low molecular weight organic compounds produced by plants, animals, and microbes, have been and continue to be a major source of new drugs and molecular probes. The majority of known natural product-derived HIF-1 activators were discovered through pharmacological evaluation of specifically selected individual compounds. The combination of natural products chemistry with appropriate high-throughput screening bioassays could provide an alternative approach to discover novel natural product-derived HIF-1 activators. Potent natural product-derived HIF-1 activators that exhibit a low level of toxicity and side effects hold promise as new treatment options for diseases such as myocardial and peripheral ischemia, and as chemopreventative agents that could be used to reduce the level of ischemia/reperfusion injury following heart attack and stroke.
PMCID: PMC2907550  PMID: 16842166
HIF-1; Natural Product; Tissue Ischemia; Therapeutic Angiogenesis; Molecular-Target; Small Molecule Activator; Chemoprevention; Ischemia/Reperfusion Injury
11.  Natural Product-Based Inhibitors of Hypoxia-Inducible Factor-1 (HIF-1) 
Current drug targets  2006;7(3):355-369.
The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates the expression of more than 70 genes involved in cellular adaptation and survival under hypoxic stress. Activation of HIF-1 is associated with numerous physiological and pathological processes that include tumorigenesis, vascular remodeling, inflammation, and hypoxia/ischemia-related tissue damage. Clinical studies suggested that HIF-1 activation correlates directly with advanced disease stages and treatment resistance among cancer patients. Preclinical studies support the inhibition of HIF-1 as a major molecular target for antitumor drug discovery. Considerable effort is underway, in government laboratories, industry and academia, to identify therapeutically useful small molecule HIF-1 inhibitors. Natural products (low molecular weight organic compounds produced by plants, microbes, and animals) continue to play a major role in modern antitumor drug discovery. Most of the compounds discovered to inhibit HIF-1 are natural products or synthetic compounds with structures that are based on natural product leads. Natural products have also served a vital role as molecular probes to elucidate the pathways that regulate HIF-1 activity. Natural products and natural product-derived compounds that inhibit HIF-1 are summarized in light of their biological source, chemical class, ancd effect on HIF-1 and HIF-mediated gene regulation. When known, the mechanism(s) of action of HIF-1 inhibitors are described. Many of the substances found to inhibit HIF-1 are non-druggable compounds that are too cytotoxic to serve as drug leads. The application of high-throughput screening methods, complementary molecular-targeted assays, and structurally diverse chemical libraries hold promise for the discovery of therapeutically useful HIF-1 inhibitors.
PMCID: PMC2908043  PMID: 16515532
HIF-1; Natural Product; Tumor Hypoxia; Molecular-Targeted Drug Discovery; Small Molecule HIF-1 Inhibitor; Hypoxia Selective
12.  Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential 
Journal of natural products  2013;77(1):111-117.
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.
doi:10.1021/np400758t
PMCID: PMC3932489  PMID: 24328138
13.  Structures and Potential Antitumor Activity of Sesterterpenes from the Marine Sponge Hyrtios communis (Carter, 1885) 
Journal of natural products  2013;76(8):10.1021/np400350k.
The extract of marine sponge Hyrtios communis (Carter, 1885) (Order Dictyoceratida, Family Thorectidae) 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 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 1, nor 2, or any of the other less active sesterterpenes suppressed breast tumor T47D or MDA-MB-231 cell viability.
doi:10.1021/np400350k
PMCID: PMC3809078  PMID: 23944963
14.  Inducers of Hypoxic Response: Marine Sesquiterpene Quinones Activate HIF-1 
Journal of natural products  2013;76(6):1175-1181.
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.
doi:10.1021/np400320r
PMCID: PMC3718637  PMID: 23731014
15.  Comparative Study of Chromatographic Medium-Associated Mass and Potential Antitumor Activity Loss with Bioactive Extracts 
Journal of natural products  2013;76(4):642-647.
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 ten extracts. Curcuma longa, Saururus cernuus and Citrus reticulata extracts showed decreased HIF-1 inhibitory activity after elution through Si gel. An additional non-polar 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).
doi:10.1021/np300858c
PMCID: PMC3683388  PMID: 23441686
16.  Semisynthetic Studies Identify Mitochondria Poisons from Botanical Dietary Supplements – Geranyloxycoumarins from Aegle marmelos 
Bioorganic & medicinal chemistry  2013;21(7):1795-1803.
Bioassay-guided isolation and subsequent structure elucidation of a Bael tree Aegle marmelos lipid extract yielded two unstable acylated geranyloxycoumarin mixtures (1–2), six geranyloxycoumarins (3–8), (+)-9′-isovaleroxylariciresinol (9), and dehydromarmeline (10). In a T47D cell-based reporter assay, 1 and 2 potently inhibited hypoxia-induced HIF-1 activation (IC50 values 0.18 and 1.10 μg mL−1, respectively). Insufficient material and chemical instability prevented full delineation of the fatty acyl side chain olefin substitution patterns in 1 and 2. Therefore, five fatty acyl geranyloxycoumarin ester derivatives (11–15) were prepared from marmin (3) and commercial fatty acyl chlorides by semisynthesis. The unsaturated C-6′ linoleic acid ester derivative 14 that was structurally most similar to 1 and 2, inhibited HIF-1 activation with comparable potency (IC50 0.92 μM). The octanoyl (11) and undecanoyl (12) ester derivatives also suppressed HIF-1 activation (IC50 values 3.1 and 0.87 μM, respectively). Mechanistic studies revealed that these geranyloxycoumarin derivatives disrupt mitochondrial respiration, primarily at complex I. Thus, these compounds may inhibit HIF-1 activation by suppressing mitochondria-mediated hypoxic signaling. One surprising observation was that, while less potent, the purported cancer chemopreventive agent auraptene (8) was found to act as a mitochondrial poison that disrupts HIF-1 signaling in tumors.
doi:10.1016/j.bmc.2013.01.048
PMCID: PMC3602229  PMID: 23434131
Botanical Dietary Supplements; Mitochondrial Poisons; Geranyloxycoumarin; Auraptene; Hypoxia-Inducible Factor-1 (HIF-1)
17.  Glycolysis Inhibitor Screening Identifies the Bis-geranylacylphloroglucinol Protonophore Moronone from Moronobea coccinea 
Journal of natural products  2012;75(12):2216-2222.
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 10,648 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.
doi:10.1021/np300711e
PMCID: PMC3532528  PMID: 23245650
18.  Structures and Mechanisms of Antitumor Agents - Xestoquinones Uncouple Cellular Respiration and Disrupt HIF Signaling in Human Breast Tumor Cells 
Journal of natural products  2012;75(9):1553-1559.
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), that 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.
doi:10.1021/np3002892
PMCID: PMC3482980  PMID: 22938093
19.  Transplanted hypothalamic neurons restore leptin signaling and ameliorate obesity in db/db mice 
Science (New York, N.Y.)  2011;334(6059):1133-1137.
Evolutionarily old and conserved homeostatic systems in the brain, including hypothalamus, are organized into nuclear structures of heterogeneous and diverse neuron populations. To investigate whether such circuits can be functionally reconstituted by synaptic integration of similarly diverse populations of neurons, we generated physically chimeric hypothalami by micro-transplanting small numbers of embryonic enhanced green fluorescent protein-expressing leptin-responsive hypothalamic cells into hypothalami of postnatal leptin receptor-deficient (db/db) mice that develop morbid obesity. Donor neurons differentiated and integrated as four distinct hypothalamic neuron subtypes, formed functional excitatory and inhibitory synapses, partially restored leptin responsiveness, and ameliorated hyperglycemia and obesity in db/db mice. These experiments serve as proof of concept that transplanted neurons can functionally reconstitute complex neuronal circuitry in the mammalian brain.
doi:10.1126/science.1209870
PMCID: PMC3770458  PMID: 22116886
20.  Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2 
Journal of natural products  2011;74(9):1894-1901.
Over 20000 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 µM 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.
doi:10.1021/np200370z
PMCID: PMC3179826  PMID: 21875114
21.  Epilepsy gene LGI1 regulates postnatal developmental remodeling of retinogeniculate synapses 
The Journal of Neuroscience  2012;32(3):903-910.
Retinogeniculate connections undergo postnatal refinement in the developing visual system. Here we report that non-ion channel epilepsy gene LGI1, mutated in human autosomal dominant lateral temporal lobe epilepsy (ADLTE), regulates postnatal pruning of retinal axons in visual relay thalamus. By introducing an ADLTE-associated truncated mutant LGI1 (836delC) or excess full-length LGI1 into transgenic mice, we found that mutant LGI1 blocks, while excess LGI1 accelerates retinogeniculate axon pruning. The normal postnatal single fiber strengthening was arrested by mutant LGI1, and contrastingly, was enhanced by excess wild-type LGI1. The maximum response of the retinogeniculate synapses, on the other hand, remained the same in mature LGI1 transgenic mice, indicating that mutant LGI1 blocks, whereas excess wild-type LGI1 promotes, weak axon fiber elimination. Heterozygous deletion of the LGI1 gene, as found in ADLTE patients, inhibited postnatal retinogeniculate synapse elimination, an effect similar to the ADLTE truncated mutant LGI1. The results identify sensory axon remodeling defects in a sensory aura-associated human epilepsy disorder.
doi:10.1523/JNEUROSCI.5191-11.2012
PMCID: PMC3342858  PMID: 22262888
22.  Thyrsiferol Inhibits Mitochondrial Respiration and HIF-1 Activation 
Phytochemistry letters  2011;4(2):75-78.
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.
doi:10.1016/j.phytol.2010.09.003
PMCID: PMC3139250  PMID: 21785662
Thyrsiferol; Hypoxia-inducible factor-1 (HIF-1); Laurencia, Triterpene polyether; Marine natural product; Mitochondrial complex I inhibitor; NADH-ubiquinone oxidoreductase
23.  Increased Gene Dosage of Ube3a Results in Autism Traits and Decreased Glutamate Synaptic Transmission in Mice 
Science Translational Medicine  2011;3(103):103ra97.
People with autism spectrum disorder are characterized by impaired social interaction, reduced communication, and increased repetitive behaviors. The disorder has a substantial genetic component, and recent studies have revealed frequent genome copy number variations (CNVs) in some individuals. A common CNV that occurs in 1 to 3% of those with autism—maternal 15q11-13 duplication (dup15) and triplication (isodicentric extranumerary chromosome, idic15)—affects several genes that have been suggested to underlie autism behavioral traits. To test this, we tripled the dosage of one of these genes, the ubiquitin protein ligase Ube3a, which is expressed solely from the maternal allele in mature neurons, and reconstituted the three core autism traits in mice: defective social interaction, impaired communication, and increased repetitive stereotypic behavior. The penetrance of these autism traits depended on Ube3a gene copy number. In animals with increased Ube3a gene dosage, glutamatergic, but not GABAergic, synaptic transmission was suppressed as a result of reduced presynaptic release probability, synaptic glutamate concentration, and postsynaptic action potential coupling. These results suggest that Ube3a gene dosage may contribute to the autism traits of individuals with maternal 15q11-13 duplication and support the idea that increased E3A ubiquitin ligase gene dosage results in reduced excitatory synaptic transmission.
doi:10.1126/scitranslmed.3002627
PMCID: PMC3356696  PMID: 21974935
24.  Natural and Semisynthetic Mammea-Type Isoprenlated Dihydroxycoumarins Uncouple Cellular Respiration 
Journal of natural products  2011;74(2):240-248.
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 fourteen mammea-type coumarins including three new compounds mammea F/BB 1 (1), mammea F/BA (2), and 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 PC3 cells (IC50 0.94 μM for hypoxia-induced). Coumarins possessing a 6-prenyl-8-(3-methyl-oxobutyl)-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.
doi:10.1021/np100762s
PMCID: PMC3045645  PMID: 21214226
25.  Mammea E/BB, An Isoprenylated Dihydroxycoumarin Protonophore that Potently Uncouples Mitochondrial Electron Transport Disrupts Hypoxic Signaling in Tumor Cells 
Journal of Natural Products  2010;73(11):1868-1872.
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 underlay 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.
doi:10.1021/np100501n
PMCID: PMC2993771  PMID: 20929261

Results 1-25 (37)