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1.  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)
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  The Marine Sponge Metabolite Mycothiazole: A Novel Prototype Mitochondrial Complex I Inhibitor 
Bioorganic & medicinal chemistry  2010;18(16):5988-5994.
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.
doi:10.1016/j.bmc.2010.06.072
PMCID: PMC2918693  PMID: 20637638
Hypoxia-inducible factor-1 (HIF-1); Marine natural products; Mitochondrial Complex I Inhibitor; NADH-ubiquinone oxidoreductase
9.  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
10.  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
11.  Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents 
The Journal of Clinical Investigation  2009;119(8):2291-2303.
The anorexigenic neuromodulator α-melanocyte–stimulating hormone (α-MSH; referred to here as α-MSH1–13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of α-MSH1–13 maturation and inactivation is incompletely understood. Here we have provided insight into α-MSH1–13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of α-MSH1–13, producing α-MSH1–12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where α-MSH1–13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of α-MSH1–13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet–induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active α-MSH1–13 levels.
doi:10.1172/JCI37209
PMCID: PMC2719925  PMID: 19620781
12.  Upregulation of prolylcarboxypeptidase (PRCP) in lipopolysaccharide (LPS) treated endothelium promotes inflammation 
Background
Prolylcarboxypeptidase (Prcp) gene, along with altered PRCP and kallikrein levels, have been implicated in inflammation pathogenesis. PRCP regulates angiotensin 1–7 (Ang 1–7) – and bradykinin (BK) – stimulated nitric oxide production in endothelial cells. The mechanism through which kallikrein expression is altered during infection is not fully understood. Investigations were performed to determine the association between PRCP and kallikrein levels as a function of the upregulation of PRCP expression and the link between PRCP and inflammation risk in lipopolysaccharide (LPS)-induced endothelium activation.
Methods
The Prcp transcript expression in LPS-induced human umbilical vein endothelial cells (HUVEC) activation was determined by RT-PCR for mRNA. PRCP-dependent kallikrein pathway was determined either by Enzyme Linked ImmunoSorbent Assay (ELISA) or by biochemical assay.
Results
We report that PRCP is critical to the maintenance of the endothelial cells, and its upregulation contributes to the risk of developing inflammation. Significant elevation in kallikrein was seen on LPS-treated HUVECs. The conversion of PK to kallikrein was blocked by the inhibitor of PRCP, suggesting that PRCP might be a risk factor for inflammation.
Conclusion
The increased PRCP lead to a sustained production of bradykinin in endothelium following LPS treatment. This amplification may be an additional mechanism whereby PRCP promotes a sustained inflammatory response. A better appreciation of the role of PRCP in endothelium may contribute to a better understanding of inflammatory vascular disorders and to the development of a novel treatment.
doi:10.1186/1476-9255-6-3
PMCID: PMC2639534  PMID: 19171072

Results 1-12 (12)