Antifouling peptides from the marine sponge Geodia barretti

Transcription

1 Antifouling peptides from the marine sponge Geodia barretti Martin Sjögren Department of Medicinal Chemistry Division of Pharmacognosy,, Uppsala University

2 Why is it important to protect a surface? Fouling cause exhaustion of materials (corrosion( corrosion) increased friction and fuel consumption increased weight

3 atural products as antifouling agents Repelling compounds, swimming away Photo: : Tomas Lundälv, TMBL

4 Initial questions as Geodia barretti a chemical defence? If so, is it possible to isolate and characterise the active compounds? Which are the target organisms? Mechanism for the compounds?

5 Advantages using cyprid larvae as a model organism experiments on a whole animal are cultivated at Sven Loven centre for Marine Science (Tjärn rnö) ) all year few neurons-more easy to study comparing to mammaliesystems receptors: dopamine, serotonine octopamine

6 ow it started; Et-extract from G. barretti % etanolextrakt av G. barretti Effect of ethanolextract Response (%) Response% K Koncentration (mg/ml) Concentration (mg/ml) Settlement% Dead % K

7 Modified extractionsprotocol from Claeson et al, 1996 Geodia barretti tissue; homogenize and freeze dry Pre-extraction with dichloromethane Removal of lipohilic contituents G. barretti residue Dichloromethane extract Extraction with ethanol (50%) Extraction of peptides Size-exclusion chromatography on Sepahdex G 10 Ethanol extract Removal of low-molecular weight compounds igh-molecular weight fraction Low-molecular weight fraction Solid-phase extraction on RP-18 material Removal of salts and polysaccharides Peptide fraction Salts and polysaccharides

8 Bioassay-guided isolation and characterisation Extraction Fractionation: : igh Performance Liquid Chromatography - PLC Molecularweight and fragmentation: Mass Spectrometry - MS Structure analysis: uclear Magnetic Resonance - MR

9 Chemical structure of the barettins isolated and characterised from G. barretti Br 2 Barettin MW:419/421 Br 2 8,9-dihydrobarettin MW:421/423 Antifouling activity of brominated cyclopeptides from the marine sponge Geodia barretti, Sjogren, M et al JURAL F ATURAL PRDUCTS: 67, 3 Pages:

10 Effect of natural and synthesised barettin on B. improvisus cyprids Br 2 barettin (natural) barettin (synthesised) Respons (%) Settlement % Living larvae % Dead larvae% Respons (%) Settlement % Living larvae % Dead larvae% 0 Control 0.15 µm 0.3 µm 0.6µM 1.2 µm 2.4 µm 24 µm 0 Control µm 0.24 µm 2.4 µm Treatment Treatment Inhibition of settlement at 2.4 µm M (0.1 µg/ml) EC 50 = 0.9µM M (0.2 µg/ml) TBT: LC 50 = 90 ng/ml

11 Br 2 Effect of natural and synthesised 8,9-dihydrobarettin 8,9-dihydrobaretti (natural( natural) 8,9-dihydrobaretti (synthesised( synthesised) Response (%) Settlement % Living larvae% Dead larvaer% Response (%) Settlement % Living larvae% Dead larvae% Control 1.0 µm 10 µm 100 µm Treatment 0 Control 1 µm 10 µm 100 µm Treatment Settlement inhibitionering between µm M ( µg/ml) Ec = 8µM 8 comparing to barettin 0.9µM

12 Bioassay-guided fractionation,, isolation and structure elucidation of the dipeptide barettin-plc MASS LC-MS of barettin RT: SM: 7B L: 1.04E7 Base Peak m/z= MS Gb ink vaten Barettin MW:419/421 EC 50 =0.9µM EC Br 2 Mass fractionation of barettin 17= 3 42=C : 59=C 2 5 +: a #1-53 RT: AV:53 L: 9.50E6 T: + c Full ms [ ] e c 75 n a 70 d n A bu 55 e v a ti 40 l e 35 R Time (min) / / / m/z Antifouling_ovotel

13 Synthesis of barettin Why synthesise? 1. The environmentally aspect: devastation of G. barretti is not an option 2. Confirmation of the substance: without a synthesis You are never sure of the right structure 3. Economical aspects (unfortunalety barettin was very expensive to synthesise)

14 Synthesis av barettin Johnson AL et al, TETRAEDR,2004 (60)

15 Analogs of barettin and dipodazine Table of activity Analogs-structures structures Substance MW EC 50 p-value F-value *(I)/(S) ( M barettin (1) I 8,9-dihydrobarettin (2) I 5-bromobarettin (3) S debromobarettin (4) 342 * dipodazine (5) bromodipodazine (6) I 5-methoxydipodazine (7) nitrodipodazine (8) chlorodipodazine (9) methyldipodazine (10) benzo(e)dipodazine (11) I 3-[1-benzothiophen-2-yl-methylidene] piperazine-2,5-dione (12) 3-[1-(6-bromo-1-indol-3-yl)-meth I (E)-ylidene]-hexahydro-pyrrolo[1,2- a]pyrazine-1,4-dione (13) 3-[1-(6-bromo-1-indol-3-yl)-meth- (Z)-ylidene]-hexahydro-pyrrolo[1,2- a]pyrazine-1,4-dione (14) bromo 1-indole-3-carboxaldehyde I X Y X Y 1 X =, Y = Br 3 X = Br, Y = 4 X =, Y = 5 X =, Y = 6 X = Br, Y = 7 X = Me, Y = 8 X = 2, Y = 9 X =, Y = Cl 10 X = Me, Y = 2 Br 11 Br 13 E isomer 14 Z isomer 2 Br S 2 (15) benzo(g)dipodazine (16) I Sjogren et al 2006: PEPTIDES, (27)

16 Fieldexperiments - results Efect on Balanus improvisus Effect on the bluemussel, Mytilus edulis Reduction of recrutiment (% of controls) SPF FabiEco barettin 8,9-dihydrobarettin Paints 2000 TF Reduction of recrutiment (% of controls) SPF barettin 8,9-dihydrobarettin FabiEco 2000 Paints TF 0.1 % of barettin in the SPF-coating resulted in a 80% reduction of recruitment of B. improvisus and a 80% reduction of M. edulis over an eight-week period Sjogren M, et al, BIFULIG, 2004 (20)

17 Mechanism Barettin (1) Br 5-hydroxytryptamine 2 8,9-dihydrobarettin (2) Br Tegaserod 2 C 2 C C C 3 Barettin and 8,9-dihydrobarettin have specific activity on 5- T-receptors-evolutionary defence? Barettin has activity on 5-T 2a 2c and 5-T 4 8,9-dihydrobarettin på p 5-T 2c (G-protein coupled receptors) edner E, Sjogren M,et al, JURAL F ATURAL PRDUCTS; 2006 (69)

18 Post-doc-project -project Mainhypothesis; Associated bacteria is the producer of the barettins PCR Cultivating on dishes,, aerobt/anaerobt DA-library Phylogeny-shikimic shikimic-acid passway (animals can not produce the aromatic amino-acids acids tryptophane, thyrosine and phenylalanine by theirselves Antifouling_ovotel

19 The future apple Receptor -ligand- studies at the barettins and examine agonism/antagonism apple Chemical diversity; similar compounds in other reef-building marine sponges; G. macandrewii,, G. atlantica and Isops phlegrae (ICES.dk dk, International Council for the Exploration of the Sea ) apple Synergy-studies of the barettins apple ther applications i.e. drugs

20 Thanks to ans Elwing and coworkers Professor Lars Bohlin, dep of Pharmakognosy,, Uppsala University Professor Per Jonsson, Marine Ecology, Sven Loven centre for Marine Science Dr Ulf Göransson, dep of Pharmakognosy, Uppsala University Dr Mia Dahlström, Dep of Fishery,, Göteborg G Professor Jan Bergman, ovum,, KI, Stockholm Dr Ann-Louise Johnson, ovum,, KI, Stockholm