Test 1 Study Guide
Test 1 Study Guide MSC462
Popular in Marine Biomedicine
Popular in Marine Science
This 19 page Study Guide was uploaded by Zack Bauman on Wednesday February 18, 2015. The Study Guide belongs to MSC462 at University of Miami taught by Dr. Liza Merly in Fall. Since its upload, it has received 272 views. For similar materials see Marine Biomedicine in Marine Science at University of Miami.
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Date Created: 02/18/15
Zack Bauman MSC 462 21715 Test 1 Study Guide Lintroduction to Marine Biomedicine Marine Biomedicine Focus 0 Drugs or potential drugs 0 Clinical trials from phase 1 through approval 0 Identifying mechanisms of action MOAs o Diverse chemical structures and taxonomic groups 0 Distinguishing microorganism or macroorganism Synergistic relationships Organism itself or endosymbionts inside foreshadow Difficult to distinguish whether compound came from cnidarian or bacterium living within 0 Secondary Metabolites Something the body produced that does not have physiological role in body Biomedicaltoolsreagents Biomedical research models 0 Natural Products vs Clinical Medicine 0 Natural Products Large component of traditional medicine Antiinfective and anticancer drugs Semisynthetics lead cancer research 0 Natural products and synthetics follow 0 Marine Natural Products MNPs Began in 19605 First isolation of spongothymidine and spongouridine from marine sponge Tethya crypta l nucleosides 0 Led to rst marine antiviral and anticancer drugs 0 Cytarabine antileukemia agent approved by FDA in 1969 Vidarabine antiviral agent approved by FDA in 1976 Next 30 years increased discovery of MNPs exponenUaHy Next FDA approval in 2004 o Ziconotide Piralt from cone snail approved in 2004 Trabectedin Yondelis from colonial tunicate in 2007 Major hindrances in marine drug discovery 0 Difficult to obtain large quantities As development of drug progresses larger amounts are needed Became an issue in drug being developed from isolate in tunicate Aquatic movement of species 0 Marine is more costly than terrestrial research Pharmaceutical companies not prepared to take the economic risk 0 Safer to stay the terrestrial course where there are some drugs quotnot completely masteredquot 0 Ocean is vast Combinatorial methods 0 Using porecreating proteins to administer other MNPs o Synergistic effects of multiple analgesicsanticancersetc Clinical Trials Occur in phases regulated through the FDA very strict process 0 Preclinical Phase soationcharacterization Mechanism of Action In vitro testing Animal testing 0 Phase 1 Looking for safest dose Most effective way to deliver Orally intravenous etc Side effects 50 or more of drugs don t make it past phase 1 2080 participants Several month process but with 70 success rate cancerspeci c stat 0 Phase 2 Effectiveness on particular group of people with speci c disease 100300 participants much more optimistic larger group willing Up to 2 years Ef ciency studied 33 success rate 0 Phase 3 Extremely large group 10003000 participants 14 years 0 Studies safety ef cacy and dosing 2530 success rate 0 Phase 4 Thousands of participants 1 year Longterm effectivenesscost effectiveness studied 7090 0 Launch 2 Marine Source of Bioactive Compounds 0 Include many different types of compounds that exhibit bioactivity pro les 0 Drug may not be good if it has more than one primary func on 0 Want highly speci c drug MOAs Secondary metabolite Organic compounds that are not directly involved in the normal growth development or reproduction of an organism 0 It s loss may result in decreased survivability or fecundity o Interspecies defense 0 Derived into MNPs TVpes of secondarv metabolites o Alkaloids Small heavily derived amino acids Morphine Analgesic Derived from plant Tetrodotoxin TTX Toxic Several applications One of the biggest groups of secondary metabolites that have been developed as MNPs o Terpenoids Largest group of natural products Lipids in nature Includes the steroids 0 Glycoside have carbohydrate component heavily modi ed sugars o Nucleosides glycosamine that consists of a base bound to a sugar 0 Polyketides Discodermalide from sponges Methods of extraction Selective extraction particular solvent Filtration lter by molecular size Precipitation Dialysis put in positively charged buffer to remove nega veions Centrifugation gravitypressurebased strati cation Open column chromatography Ion exchange Size exclusion Af nity chromatography Salting out 0000 CO FDA Approved Drugs 0 Cytarabine Cytosar U Anticancer Based on spongothymidine nucleoside Cytotoxic agent because it competes intracellularly with substrates for various enzymes involved in DNA Elm Inhibits DNA polymerase and DNA synthesis Causes suppressed immune system 0 Vidarabine ViraA Antiviral Based on spongouridine nucleoside Inhibits viral DNA polvmerase and SNA synthesis of herpes vaccinia and other virals etc Topical application No longer being marketed because better antivirals have become popular 0 Ziconotide Piralt Analgesic Based on conotoxin isolated from cone snail venom Analgesic pain reliever Cox 2 inhibitor inhibition to relieve pain Reversiny blocks calcium channels located in afferent nerves in the spinal cord reducing release of neurotransmitters Administered intrathecally l directly into spaces in spinal column Severe pain killer comparable to Morphine o Yondelis Currently being developed in US Already approved for number of cancers in EU EU slightly less strict Semisynthesis now well established Highly cytotoxic Binds to DNA minor groove and interacts with different binding proteins of the nucleotide excision repair system and stops GZM cell cycle Being developed for breast lung prostate and pediatric cancer Phase 3 drugs 0 Number of isolates and synthetics thereof derived from sponges precancer o Eribulin mesylate Currently in multiple clinical trials lf tubulin impaired Cell membrane impaired Cell can t divide cytoskeleton Endocytosisexocytosisimpaired Water soluble and chemically stable l taken orally Phase 2 drugs 0 DMXBA Used for schizophrenia Comes from marine worm Based on alkaloid present in marine worms Simulates acetvlcholine receptors expressed on CNS neurons astrocytes and peripheral macrophages lmproves cognition and de cient sensory gating in animal models o Many phase 2 anticancer drugs Tunicate isolates Extremely potent inducer of apoptosis Apoptosis programmed cell death normal part of the cell s life Necrosis Cell falls apart membrane falls apart and things leak out could be toxic to other cells Elisidepsin o Antitumor activity MOA unknown PM00104 DNA binding alkaloid isolated based on isolates from skin and mucus of tunicates and sponges cancel cell apoptosis Phase 1 druos Marizomib Anticancer from Salinispora tropica o Potent Selective inhibition of proteasome complex Bryostatins 0 Marine drugs with anticancer effects from Buguta neritina Bryostatin 1 Derived from Bryostatin Bind to protein kinase C 0 Part of signaling cascade telling cells what to do 0 In ammation immune responses and intracellular signaling LSponges as Pharmacy Then and Now Over 10000 compounds isolated as marine natural products Sponges o Sessile 0 Most primitive metazoan Rudimentary immune defenses o Phagocytosis 0 Only innate components of immune response and relatively few 0 Exposure to surrounding water environment at cellular level 0 Filterfeeding o Organisms have to protect themselves because these animals are constantly exposed to microbes Ecological role for secondary metabolites 0 Emission of mucuscontaining toxins protection 0 Isolated compounds including bacteria algae fungi bryozoa corals tunicates and other organisms o No other compounds showing quite as structurally complex than those derived from sponges o No true tissues multicellular 0 Water ows through porous body and out of the osculum Ecological role of sponge metabolites 0 There are a high number of the compounds and they are extremely diverse and complex Biofouling prevention presents ecological advantage 0 Benthic marine organisms constantly affected by settlement of larvae propagules and microorganisms on their surface 0 Habitats with high level of spatial competition Coral reefs speci cally Antifouling mechanisms 0 Waterpumping biology requires that sponges be free of settlement by other marine organisms o Bio lm formation andor settlement by barnaclesbryozoans on surface 0 Antifouling agents released to minimize this colonization Azidothymidine AZT AntiHIV 0 Type of antiretroviral drug 0 Inhibits the reverse transciptase enzyme that HIV virus uses to svnthesize DNA prevents viral DNA from forming Three main classes of anticancer agent Nonspeci c inhibitors of cell growth 0 Speci c inhibitors of cancer cells l inducers of apoptosis Inhibitors of cancer cells of a certain type of cancer i Endosymbionts The problems and promise they present to the scienti c community Endosymbionts Broadly de ned as a permanent association between two individuals of different species Marine ecosystems contain many functionally critical symbioses True symbionts may be shielded from sponge consumption due to speci c recognition mechanisms or surface characteristics eg slime capsules What makes a symbiont 0 First genomic insight Future genomic studies of sponge symbionts will revolutionize our understanding of a range of hostsymbiont phenomena 0 Signaling between partners 0 Metabolic interdependencies 0 Possible symbiont genome reductiondiversi cation Spongemicrobe symbioses lead to symbiotic relationships with their hosts 0 Advantages to the host 0 Nutrition 0 Transportation of chemicals throughout sponge tissue 0 Assistance in chemical defense and removal of waste products from sponge 0 Structural rigidity Using the microbes as overall structure 0 Complexity of spongesymbiont relationship 0 Many different bacterial species permanently inhabit sponges 0 Contribute to total sponge biomass Some examples 0 Cyanobacterial symbionts can contribute up to 40 of the sponge tissue Produce many active compounds including anti microbials 0 Host sponge likely bene ts because it keeps the sponge free of other bacteria 0 Three broad types of microbial associates in sponges o Abundant populations of spongespeci c microbes in the sponge mesohyl 0 Small populations of speci c bacteria occurring intracellularly Speci c enough to one side of a reef on the species ofsponge o Populations of nonspeci c bacteria resembling those in the surrounding seawater These are the ones that appear to be transient and are similar to the surrounding seawater that the spongeisfoundin 0 Example 0 One type of bacterial isolate was recovered from 35 different sponges but not found in the seawater Why can this symbiont be such a good symbiont And why does everyone want that symbiotic relationship 0 Immunological experiments in which theses same isolates crossreacted with other spongespeci c bacteria but not with seawater isolates were taken as further evidence of sponge speci city 2 big questions 0 Where do they come from 0 Vertical transmission and environmentally acquired is the main hypothesis 0 Vertical transmission and environmentally impacted separately are other hypothesis but they seem to be ruled out o How are they maintained 0 Less is known about the maintenance of the symbionts Future research in sponge microbiology Accessing bioactive compounds from sponge symbionts by metagenomics and cultivation strategies 0 De ning the members of the stable versus transient microbial communities 0 Can be very stable in a petite subbiome 0 Could be found in different species around the world with many varying parameters Coevolution of sponges and microorganisms symbioses by de nition Physiology of sponge symbionts Hostsymbiont interactions l signaling Usefulness to bioactive compound search and discovery Obvious bene t to drug development from bioactive compounds 0 Beyond that bene t of studying oldest metazoanmicrobe symbioses 0 Insight into microbiomes in higher animals and their role in overall physiology and health Localization of metabolites cell separation techniques 0 First establish which cell types are responsible for metabolic activity Separation techniques Sponge tissue can be easily disassociated Use of ow cytometry to separate sponge cells from their cyanobacterial symbionts and other associated microbes 0 Sample cells mixed when going into the ow cytometer in suspension Put into ow cell one after another in the pathway Arranged in a line moving single le Laser light source strikes sample as it crosses the path Measure light scattered forward and side scatter 0 Based on auto uorescence Separation due to tissue size and density 0 Sponge cells 0 Unicellularcyanobacteria o Unicellular heterotrophic bacteria 0 Filamentous heterotrophic Early evidence suggested that both host and symbiont are sources of bioactive compounds 0 Three major types of known metabolites from this sponge 0 Two found exclusively inside 0 spongeiae cells 0 One was con ned to sponge cells Supply issue Cultivation of relevant microbe sponge culture or expression of biosynthesis genes 0 Relevant microbe must parallel usual parameters ie temperature acidity etc o Sponge culture staining used 0 Biosynthesis used with other commonly used bacteria e coli for example 0 Challenge of ensuring continuous largescale production of bioactive compound for clinical trials and continuous supply to the market 0000 LWW bioactive compounds and syntheti pathways the wave of the future Bacterial MNP as important course of novel structures for drug discovery 0 Secondary metabolites Cytotoxic properties 0 Advances in marine molecular biology genomic sequencing bioinformatics Biosynthetic pathways can be exploited to design compounds Phytoplankton 0 Mostly unicellular 0 Generally very small virtually invisible o Encompass all 3 of taxonomic domains archaea bacteria and eukarya 0 Prokaryotic Cyanobacteria 0 Marine photosynthetic bacteria blue green algae o Curacin A isolated from bluegreen algae Binds to tubulin which disrupts microtubule assembly Putative anticancer agent Antimitotic activity Problems 0 Low water solubility 0 Lack of chemical stability Preclinical and clinical trials Unique structure that requires PKSlike pathway 0 Stromatolites 0 Fossilized structures left behind by cyanobacteria 0 Ubiquitous in world s oceans Source of many products 5 tropica genome Fully sequenced in 2007 0 Complex secondary metabolome 10 of genome dedicated to natural product assembly 0 High diversity of PKSs NRPSs or a mix 0 Enzymatic activity 0 Produce many diverse compounds 0 Strain harbors at least 19 biosynthetic loci Encode a broad variety of natural product classes Siderephores Polyketides Melanins Nonrobisomal and ribosomal peptides o Terpenoids Salinosporamide A 0 Highly potent irreversible inhibitor of the 205 proteasome o Synthesized through biosynthetic pathway 0 Ongoing clinical studies rely on largescale saline fermentation for continuous drug supply 0 Potent proteasome inhibitor entered clinical trials only 3 tears after its initial discovery Antiangiogenic factors derived from marine fungi Toluquinol 0 Product produced by marine fungus o Antiangiogenic activity 0 Stopping the formation of new blood vessels 0 Treatment of activated endothelial cells resulted in strong inhibitory effect 0 Could be due to induction of apoptosis 0 Both in vitro and in vivo effects observed Red tide toxins and antitoxins Cyanobacteria shown to produce the antitoxin as well as toxin 0 Brevenal brevetoxin may have therapeutic effects in the treatment of cystic brosis and other mucociliary diseases Patented in 2007 0000 iAntimicrobial Peptides from Diverse Marine Organisms Include Enzymes such as lysozyme Cathepsin G Phospolipase A2 T ransferrin Antibacterial proteinspeptides Diverse body uid factors Lysozyme ubiquitous in body tissues uids Saliva Mucus produced by the skin or other epithelial cells Antimicrobial proteinspeptides AMPs Rapidly kill broad spectrum of microbes 0 Located within granular immune cells and on epithelial layers Disrupt microbial membranes 0 Identi ed within Porifera Produced in both unicellular and multicellular organisms Crucial part of the innate immune system Participate in neutralization and elimination Potential of AMPs Gotten increased attention over last few decades 0 Natural antibiotics Promising role as therapeutics or drug leads Emergence and increase of bacterial strains resistant to antibiotics o NEED for supplementary antimicrobials Development of new antibiotics focused on modi cations of already existing pharm May improve ef ciency and potency but exposes bacteria to limited arsenal M RSA AMPs Peptides between 1 and 12 kDa Up to 2025 amino acids in length 0 Exhibit profound activity against microorgansisms in vitro Coded by single genes and ribsomally synthesized Different from strange modi ed cyclical peptides synthesized by mircrobes MOA of AM Ps 0 Most AMPs share two features that allow them to interact with microbes 0 They have a net positive charge which enables them to interact through electrostatic forces with bacterial membranes The membrane is anionic 0 They can form an amphipathic structures in hydrophobic environments Can penetrate into bacterial phospholipid bilayer Bacterial membrane contain more acidic phospholipids which hold a more stable more negative charge so they interact well with microbes MOA divided into two main categories Peptides that bind to bacterial membrane and create irreversible pores or destabilize membrane causing ef ux of cytoplasm Peptides that cross bacterial membrane and inhibit growth by binding intracellular components Membranenondisruptive antibacterial action 0 Strategy is less prone to cause release of endotoxins important for host Considered more selective towards microorganisms Membrane proteins represent fundamental structure in cell 0 In either MOA rst step is interaction with bacterial membranes or membrane components Bacteria less likely to develop resistance 0 Target fundamental structures than conventional antibiotics AMPs from invertebrates Crustaceans are insects of the sea Rely on innate immune components for defense 0 Habitat speci c adaptations of AMPPs are likely to exist 0 Expression of AMPs in insects and crustaceans differ markedly Insects vs Crustaceans Insects activate AMP production after bacterial exposure Crustaceans express AMPs constitutively 0 Number of AMP isolated from terrestrial invertebrates Marine invertebrate AMP defenses 0 Marine environment remains largely unexplored 0 Number of diverse habitats with lots of molecules for defense against predators pathogens etc 0 Marine animals are in close proximity to microbes density of 105 107 mL 0 40 different AMPs characterized 0 Only a few belong to AMP family already found in terrestrial Diversity in sequence Peptides diversi ed through evolution given diversity of habitats and microbe interactions 0 Single species can produce an array of different AMPs Differentiated AMP spectrum 0 Multiple agents acting on various targets 0 Different AMPs act complementary synergistically separately etc o In Mollusk o Mytimycin antifungal o Defensins and Myticins gram and bacteria 0 Mytilins broad activity with several isoforms Isolation and characterization Difficulties in obtaining sufficient quanitites of bioactive peptides Dissect organisms into different tissuesorgans and body uids before extraction 0 Also provides indication of whether animal produces active peptide itself or if it is derived from diet or associated parasites or microbes Actinoporins explained in paper on next page 7 Actinoporin amp Coral Papers Sea anemones sedentary animals that rely on toxins to capture and digest prey as well as an important defense from predation Among various toxins cytolytic proteins actinoporins are a very important component of venom which is very potent Actinoporins Action Target cell membranesform pores causing cell lysis and death Speci cmake pores only in membranes that contain sphingomyelinkind of lipid Interest Used as model proteins to study protein membrane interactions speci c recog of lipids etc o 2 most studied Equinatoxin ll Stycholysin II o Actinoporins not only found in sea anemones actinoporinlike proteins found in 3 animal phyla and 2 plant phyla Structural similaritvB foldused for speci c binding equivalent amino acids in binding site indicate same mechanism of binding binding site located on same side of sandwich Evidence Toxin not active against bacterial spheroblasts which lack sphingomyelin in membranes Equitine didn t form pores in phospatidylcholine lipid membrane without presence of sphingomyelin Sticholysin had binding sites for phosphocholine head group of sphingomyelin Study showed toxin didn t form pores in liposomes containing sphingolipids from sea anemone only worked when sphingomyelin added to liposomes Why protein doesent act against sea anemones membranes Anemones contain large proportions of phosphonosphingolipids which have a diff phospholipid head group makes toxin act speci cally on prey membranes that contain sphingomyelin Actinoporin also make pores in phosphatidychoine membranes containing colesterol Why Induces microdomain formationalters phosphorylcholine group making it accesible for toxin binding Hypothesis that eq ll recognises sphingm tested using Direct binding assays Mutagenesis Lipid dot bb assays Surface plasman resonance experiments Showed eq speci cally bind sphingomyelin or its analogue but not other lipids particularly phosphatidylcholine or colesterol Phosphatidylcholine and sphingomyelin have same phosphyl choline head group toxin needs to recognise sphingomyelin elsewhere in region below headgroup with Trp 112 and Tyr 113 side chains imp for further lipid recognition Actinoporin formation is a multistep process 1 Toxin recognized sphingomyelin using aromatic rich region and POC binding site attaches to membrane 2 Nterminal segment is transferred to lipid water interface 3 Toxin oigomerises on the surface of the membrane alpha helices 4 3 or 4 monomers insert into the membrane 5 N terminus is inserted through membrane to form ion conductive pathway pore Relates to MSC 462 Sponges Mucus vs actinoporin toxins for protection from predation Same potential use for the actinoporin for speci c cancer niches Piralt o Extraction of toxin for very speci c and powerful analgesic like Piralt Coral Antiin ammatory effects Arthritic counter one cora was isolated from sinuaria and was found to be potent in treating adjuvantinduced arthritis o It inhibits the production of pro in ammatory proteins iNOS and Cox 2 in murine macrophages Anticancer properties Sinularian again was a new inhibitory 50 of tumor growth in cell lines 0 Down regulation effects with cytotoxins and apoptosis production Potential antiviral properties 0 Very effective on a variation herpes virus Hard tissue therapy Naturally occurring calcium in coral aragonite and calcite will assist with protecting against and reversing bone loss when used with zeolite Scientists are looking into using the bone repair method for human teeth Neuroprotection Parkinson s disease treatment 0 Associated with in ammation and apoptosis 0 11dehydrosinulariolide Blocks two in ammatory pathways iNOS and Cox 2 Neuropathic pain reducer Analgesics o Austrasulfone iNOS and Cox 2 Other coral uses Coral sand can be used as a silicon source to reduce blood pressure Hypertensive rats can be used to improve the expression of genes that increase cardiovascular health Criticism Too little information on the effects of the drugs 0 Where the drugs are in clinical trials 0 Little to no use of the graphs
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