Discovery of Small-Molecule Natural Products That Target Cellular Bioenergetics
Total Page：16
File Type：pdf, Size：1020Kb
University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 1-1-2012 Discovery of small-molecule natural products that target cellular bioenergetics Sandipan Datta University of Mississippi Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Datta, Sandipan, "Discovery of small-molecule natural products that target cellular bioenergetics" (2012). Electronic Theses and Dissertations. 1500. https://egrove.olemiss.edu/etd/1500 This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. DISCOVERY OF SMALL-MOLECULE NATURAL PRODUCTS THAT TARGET CELLULAR BIOENERGETICS A Dissertation presented in partial fulfillment of requirements for the degree of Doctor of Philosophy in the Department of Pharmacognosy, The University of Mississippi by SANDIPAN DATTA December 2012 Copyright Sandipan Datta 2012 ALL RIGHTS RESERVED ABSTRACT Molecular-targeted antitumor therapy has found favor in antitumor drug discovery programs. Hypoxia (< 5% oxygen) is a common feature of solid tumors and hypoxia-inducible factor-1 (HIF-1) represents an important antitumor target. Bioenergetic homeostasis is typically altered in tumor cells and HIF-1 plays an important role to produce a glycolytic phenotype. Glycolysis inhibitors are an emerging class of potential tumor-selective adjuvant therapeutic agents. Natural product aerobic glycolysis inhibitors may enhance the effectiveness of current therapies. Mitochondrial oxidative phosphorylation inhibitors in botanical dietary supplements (BDS) possess a potential health hazard which should be identified and appropriately regulated. Chapter one briefly reviews the molecular-targeted natural product antitumor drug discovery process. Descriptions of tumor hypoxia, HIF-1, HIF-1 regulatory pathways and the effect of HIF-1 on tumor cell bioenergetics are presented. Cellular bioenergetic pathways and natural products that inhibit HIF-1 by interfering with cellular bioenergetics are discussed. Mitochondriotoxic small-molecule natural products reported previously are further reviewed. Chapter two discusses the effect of chromatographic media on molecular-targeted antitumor drug discovery. A panel of crude extracts was eluted through columns of various chromatographic media using a step gradient method. Total recoveries from the various columns and various elution protocols were compared and statistically analyzed. The crude extracts and column eluates were evaluated for HIF-1 inhibitory activity. Chapter three discusses the development of a bioenergetics-based screening method to screen crude extracts for glycolysis inhibitors. Crude extracts (10,648) were screened and seven ii hits (hit rate 0.72%) were identified. Bioassay-guided isolation of Moronobea coccinea crude extract resulted in isolation of a protonophoric compound moronone (1) (false positive). The structure of 1 was determined by a combination of spectroscopic and spectrometric means. The protonophoric compounds must be rapidly dereplicated for successful discovery of glycolysis inhibitors. Chapter four discusses the screening of BDS products and pure compounds for mitochondrial uncouplers or electron transport chain inhibitors. The blue cohosh (Caulophyllum thalictroides) extract and three saponins cauloside A (3), saponin PE (4) and cauloside C (5) permeabilize the mitochondrial membrane. Sesamin (6) and guggulsterol III (7) and guggul (Commiphora wightii) extract inhibit mitochondrial complex I. These extracts and compounds are cytotoxic in nature. iii DEDICATION This dissertation is dedicated in memory of my grandmother, Parul Datta, who constantly nurtured my early education and inspired me to be a better human being. iv LIST OF ABBREVIATIONS 1,3-BPG 1,3-bisphosphoglycerate 17AAG 17-N-allylamino-17-demethoxygeldanamycin 2-DG 2-deoxy-D-glucose 2-PG 2-phosphoglycerate 3BrPA 3-bromopyruvate 3-PGA 3-phosphoglycerate 4E-BP Eukaryotic initiation factor 4E-binding protein 6PP 2'-4'-dihidroxy-5'-(1'''-dimethylallyl)-6-prenylpinocembrin AcCoA Acetyl coenzyme A ADP Adenosine diphosphate ALD Aldolase ALDA Aldolase isoform A ANOVA Analysis of variance Apaf Apoptotic protease activating factor ARNT Aryl hydrocarbon receptor nuclear translocator ATCC American type culture collection ATP Adenosine triphosphate BCA Bicinchoninic acid BDS Botanical dietary supplement bHLH basic Helix-loop-helix v BNIP Bcl-2/adenovirus E1b 19 kDa protein interacting protein CBP CREB binding protein CDK Cyclin-dependent kinase CITED p300/CBP-interacting transactivator with glutamate (E)/aspartic acid (D)- rich tail COX Cytochrome c oxidase CREB cAMP reactive element binding protein C-TAD C-terminal transactivation domain DCA Dichloroacetate DHAP Dihydroxyacetone phosphate DISC Death-inducing signaling complex DMEM Dulbecco’s modified eagle’s medium DNA Deoxyribonucleic acid EGFR Epidermal growth factor receptor EGTA Ethylene glycol bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid eIF Eukaryotic initiation factor ENO Enolase EPO Erythropoetin ERK Extracellular signal-regulated kinases ETC Electron transport chain ETF Electron transferring flavoprotein vi F1,6BP Fructose-1,6-bisphosphate F2,6BP Fructose-2,6-bisphosphate F6P Fructose-6-phosphate FAD Flavin adenine dinucleotide FBS Fetal bovine serum FCCP 2-[{4-(trifluoromethoxy)phenyl}hydrazinylidene]propanedinitrile FDA Food and Drug Administration FH Fumarate hydratase FTase Farnesyl transferase G6P Glucose-6-phosphate GA3P Glyceraldehyde-3-phosphate GAPDH Glyceraldehyde-3-phosphate dehydrogenase gCOSY Gradient 1H-1H chemical shift correlation spectroscopy GGTase Geranylgeranyl transferase gHMBC Gradient heteronuclear multiple-bond coherence spectroscopy gHSQC Gradient heteronuclear single quantum coherence spectroscopy GLUT Glucose transporter gNOESY Gradient Nuclear Overhauser effect spectroscopy GSK Glycogen synthase kinase HDAC Histone deacetylase HEP3B Human hepatoma cell line vii HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] HER2 Human epidermal growth factor receptor 2 HIF-1 Hypoxia-inducible factor-1 HK Hexokinase HPLC High performance liquid chromatography HRE Hypoxia-response element HRESIMS High-resolution electron spray ionization mass spectrometry Hsp90 90 kDa heat-shock protein HTS High-throughput screening HUR Human antigen R IR Infrared spectroscopy IRES Internal ribosome entry site KA Koningic acid LD50 Lethal dose 50% or median lethal dose LDH Lactate dehydrogenase LND Lonidamine MAPK Mitogen-activated protein kinase MCT Monocarboxylate transporter MDA-MB-231 M. D. Anderson-metastatic breast-231 human breast adenocarcinoma cell line viii MEK Mitogen-activated protein kinase kinase/ extracellular signal-regulated kinase MHz Megahertz MiR MicroRNA MMP Matrix metalloproteinase M-PER Mammalian protein extraction reagent MPTP Mitochondrial membrane permeability transition pore MTOR Mammalian target of rapamycin NADH Nicotinamide adenine dinucleotide reduced NADP Nicotinamide adenine dinucleotide phosphate NCI National Cancer Institute NCNPR National center for natural products research NDUFA4L2 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex 4-like 2 NIH National Institutes of Health NLS Nuclear localization signal NMR Nuclear magnetic resonance N-TAD N-terminal transactivation domain OD Optical density ODDD Oxygen-dependent degradation domain OXPHOS Oxidative phosphorylation PAS Period cicardian-aryl hydrocarbon nuclear translocator-single-minded ix PBD Polyprenylated benzophenone derivative PDK Pyruvate dehydrogenase kinase PDP Pyruvate dehydrogenase phosphatase PEP Phosphoenolpyruvate PER Period cicardian PERK Protein kinase RNA-like endoplasmic reticulum kinase PFKBF3 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 PFKL Phosphofructokinase isoform L PGAM Phosphoglycerate mutase PGI Phosphoglucoisomerase PGK Phosphoglycerate kinase PHD Prolyl-4-hydroxylase PI3K Phosphatidylinositol-3-kinase PIP3 Phosphatidylinositol-3,4,5-triphosphate PK Pyruvate kinase PKC Protein kinase C PKM2 Pyruvate kinase isoform M2 Plk3 Polo-like kinase 3 PPP Pentose phosphate pathway PTB Polypyrimidine binding protein PTM Posttranslational modification x Q Ubiquinone QH2 Ubiquinol qHTS Quantitative high-throughput screening RACK Receptor of activated protein C kinase RBM RNA binding protein Rbx Ringbox protein REDD Regulated in development and DNA damage responses RHRE RNA hypoxia-response element RNA Ribonucleic acid ROS Reactive oxygen species RP Reverse phase SENP Sentrin/SUMO-specific protease SIM Single-minded protein SIRT Sirtuin SSAT Spermidine/spermine-N1-acetyltransferase SUMO Small ubiquitin-like modifier T47D Human breast ductal carcinoma cells TCA Tricarboxylic acid TES N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid TK Thymidine kinase TLC Thin-layer chromatography xi TMPD N,N,N′,N′-tetramethyl-p-phenylenediamine TMRM Tetramethylrhodamine methylester TNF Tumor necrosis factor TOF Time-of-flight TPI Triose phosphate isomerase TRAIL TNF-related apoptosis-inducing ligand TRK Tyrosine kinase TSC1/2 Tuberous sclerosis UM University of Mississippi UTR Untranslated region UV Ultraviolet VDAC Voltage-dependent anion channel VDU2 pVHL-interacting de-ubiquitinating enzyme VEGF Vascular endothelial growth factor pVHL von Hippel-Lindau protein xii ACKNOWLEDGEMENT First and foremost I would like