From marine enzymes to pharmaceuticals - A presentation by Dag Rune Gjellesvik

From marine enzymes to pharmaceuticals Experiences and plans for Marine Bioprospecting Biotec Pharmacon ASA Dag Rune Gjellesvik Biotec Pharmacon’s activities • Develop pharmaceuticals that work through the innate immune system • We produce, market and sell – -1,3/1,6-glucan for animal health – -1,3/1,6-glucan as human dietary supplements – Marine enzymes for research and diagnostics • Plan to develop new pharmaceuticals based on marine bioprospecting Why marine enzymes? • Marine organisms in the Northern seas are adapted to a cold environment – The enzymatic apparatus must be adapted • Particular features by coldadapted enzymes:  Higher activity at low temperature  Easy inactivation by moderate raise in temperature  Generally higher catalytic efficiency – Suitable for processing temperature sensitive foods – Provides an ”off-button” – In many cases very useful Starting with fish processing • Enzymes were used for advanced fish processing • Developing processes where proteases were utilised in Scaling Caviar production Skinning of squid Production of Cod pepsin • Autolysis of cod stomachs: – Pepsins break down other proteins – Cells are broken down, fats are liberated and flotated, some proteins precipitate – Pepsin can be concentrated by ultrafiltration – A crude concentrate is spraydried • Result: – approx. 50 kg product from 1 ton – 5 % purity Example: production of Salmon caviar Protease High-purity enzyme: Shrimp Alkaline Phosphatase • Used as a research tool – Dephosphorylation of DNA – Removal of nucleotides from PCR products • Before sequencing • Before genotyping • Key property: completely inactivated by heat • Produced from Shrimp thawing water • Requires extensive purification – Free of DNases – Free of RNases – Free of proteases Production of SAP Reketining Foto: Frank Gregersen, Fiskeriforskning Reketinevann • Raw material – – – – 4000 L Shrimp thawing water 30 kg dry matter 1 mg/L SAP Purity 0,01% • Product: – 120 ml solution – 0,5 - 1 gram protein – Purity ~100% Recombinant marine enzymes • Cod Uracil-DNA glycosylase – DNA repair enzyme – 500 kg cod liver is required to make 1 mg enzyme – Gene for Cod UNG isolated – 7 mg produced by 1 L culture • Shrimp nuclease – A new DNase with a new specificity – Developed from a sidestream in SAP production – 1 mg from 100 L thawing water – Recombinant: 28 mg from 1 L culture Cod Uracil-DNA glycosylase • Use: PCR carry-over prevention – Removal of contaminating previous PCR products 0 E.c R E Cod • Critical property: heat lability – Completely inactivated – Irreversibly inactivated • Cod UNG the only on the market with these properties Shrimp nuclease • Initial objective – Screen our shrimp thawing water for heat labile DNase(s) – Produce another enzyme in a side stream • New enzyme, new uses – Carry-over control in PCR • pre-treatment of master-mix – Convenient removal of contaminating DNA in RT-PCR • Can be included in master-mix • Found a ”new” enzyme – Particularly specific to doublestranded DNA – Very high specific activity Pharmaceutical development • -1,3/1,6-glucan from yeast – Purified beta-glucan from yeast cell walls are biological response modifiers – Pathogen associated molecular pattern (PAMP) • Activates the innate immune system • May function as immune modulator – Anti-inflammatory • History: Moving yeast betaglucan from feed additive to pharmaceutical – Feed additive in aquaculture and animal husbandry – Adjuvant in fish vaccines – Health supplement for humans – Cosmetic ingredient (cosmeceutical) – Pharmaceutical compound Current clinical trials • • • • • Oral adm. soluble glucan Asthma / allergy Radiation protection Gingivitis / periodontitis Cancer immunotherapy enhancement • Wound healing • Oral mucositis from cancer radiotherapy / chemotherapy • • • • • Phase I finished Preclinical phase Preclinical phase Preclinical phase Phase I/II in planning • Pilot study completed (diabetes ulcers) • Phase II started Future pipeline • ”Drugs from the sea” – Natural compounds are still the primary source for new drugs – The marine environment may form a rich source for new compounds • Focus: Modulators of innate immunity – Anti-inflammatory – Immunostimulatory – Anti-infectives • Marine biobank / screening platform MarBank Marine Biobank Tissues • Mammals/ vertebrates • Invertebrates • Zoo-and phytoplankton • Corals / sponges Blood/sera Live cells Bacteria/algae Extracts (water/ organic soluble) DNA/RNA Specimen collection Cryopreservation (nitrogen/ freezers) cDNA/ gene libraries Chemical fixation • Bacteria • Algae D a t a b a s e sampling/taxonomy preparation storage data Marbio Activities Marine resources Screening Anti-bacterial Anti-tumor Follow-up • Extraction • Separation  HPLC/ FPLC Research projects Anti-viral Anti-inflammatory Lead compounds Immunostimulatory MarBio tasks Organisms and tissues Extracts DNA isolation Libraries (genomic, expression, cDNA) Genome mining (bioinformatics) Expressionbased screening Sequencebased screening DNA Microarray Identification of active proteins and genes Organic phase (small molecules) Water phase (peptides and proteins) First (primary) screening (bioassays) Separation (organic phase) Second screening (rescreening) Separation (water phase) Second screening (rescreening) Identification (LC/GC-MS, NMR, etc.) Identification (LC-MS) Low Mw compounds Peptides Proteins Proteins Sequencing facilities Labforum /AUN Synthesis and purification Cloning, in vivo/in vitro expression and purification 3D Structure determination (NMR, X-ray) Partners in MarBio • The University of Tromsø – Institute of Marine Biotechnology – Institute of Chemistry – Institute of Medical Biology • The University Hospital of North Norway – Institute of Pharmacy • The Norwegian Polar Institute, Tromsø, NPI • Biotec Pharmacon ASA, Tromsø • Probio Nutraceuticals AS, Tromsø • The Norwegian Structural Biology Centre (NORSTRUCT/UoT)