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Werner E.G. MÜLLERHeinz C. SCHRÖDER
Institute for Physiological Chemistry, Medical Center,
Johannes Gutenberg-University; Duesbergweg6; D-55128 Mainz; GERMANY.
E-mail: [email protected]
NanotecMARIN GmbH, MainzBiological Resources of the Sea:
Challenges and solutions
NanotecMARIN GmbH offers a unique technology that allows the biomimeticsynthesis of silica- one major material in nanotechnology.- including opto- and microelectronics.
Prof. Dr. W.E.G. Müller
Dr. M. Wiens
Prof. Dr. Dr. H.C. Schröder
J. Grams (lawyer)
Prof. Dr. P. Lücker(pharmacologist)
R. Scharping(former chairman
of SPD)
Prof. Dr. H. Riesenhuber(former minister of
research)
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Biosilica: 22 patents worldwide
Turnover: 600.000 €
Staff: six scientists
Own laboratories
Grant money
Venture capital money
GDF - Dental Research & Innovation (Rosbach)
Schütz Dental GmbH (Rosbach)
tons of water/day/kg
Sponges: filter feederBlood vessel
Sponges: PoriferaSource of drugs
3
bacteria: gram negative
Sponges: filter capacity1 ton/day/kg
106 ml/ton- : 106 bacteria/ml = 1012 bacteria/day
DNA length:1.5 x 106 km
=20 x to moon and back
Length of bacteria:1000 km long
=Length of Germany
Sponges: PoriferaSource of drugs
Sponges: richest source for secondary metabolites
Porifera: bioprospecting
Bioactivity per phylum
relativecytotoxicity
number ofextracts tested:
Porifera 1,041
Bryozoa 56
Chordata 263
Cnidaria 395
Echinodermata 154
Mollusca 162
Crustacea 40
Rhodophyta 179
Chlorophyta 83
Phaeophyta 100
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Chemical „Blind Cow“ Game: combinatorial chemistry
Activity trials – synthesis – screening
5.000 to 10.000 substances
Activity screening – synthesis – screening
~ 200 substances
Clinical phase I
10 substances
Clinical phase II
8 substances
3 years
3 years
2 years
3 years
Combinatorial chemistry
[5 - 10,000 chemicals synthesized]
200: pre-clinics
10: clinical phase I
phase IIphase III
~1: registration
proof of concept
Biological Selection
BIOTECmarin
Natural selection and optimization
5.000 to 10.000 substances
Challenge:
discovery of the hits
Activity of biomedical interest
5.000 to 10.000 hits
Evochemistry
[200 secondary metabolites]
natural constraints and selection:
200: pre-clinics
target finding
>> 10: clinical phase I
phase IIphase III
>> 1: registration
proof of concept
5
Memantine
Flupirtine
6
Avarol
araA
Vidarabin
1975:first drug
in medical use
alpha beta RSV HSV0
20
40
60
80
100
DNA polymerase
Km
Ki
Sponges: PoriferaSource of drugs
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H.J.Breter – Universität MAINZProf. Dr. W. Geurtsen – Med. Hochschule HANNOVER W.E.G. Müller – Universität MAINZP. Proksch – Universität DÜSSELDORFH.C. Schröder – Universität MAINZDr. Matthias Wiens – Universität MAINZProf. Dr. Wolfgang Tremel – Universität MAINZR. Batel – Rudier Boskovic Institute HR-52210 ROVINJ
BiosilicatBiofouling
Spinn off company:
BIOTEC marin GmbH
Molecular marine biotechnologyof marine sponges and theirassociated microorganisms
Coordination:W.E.G. Müller – Universität MAINZ
Center of Excellence
J.W.-H. Li, J.C. Vederas (2009)Drug Discovery and Natural Products: End of an Era or an Endless Frontier? Science 10 July 2009; pp. 161 - 165
Marine sources of drugs
Number of drugs approved in the United States from 1981 to 2007
8
chemical
physical
PKS-3_SUBDO MSTREARLMDPQHRLLLEMAWRA VEHSGTAPSALANTNTGVFVGLATH DYLGIGLWLLTYPEIEAYLAIGTP NAAAA 77PKS-5_SUBDO ISPREAAEIDPQQRMLLEMSWQC MEDAATGADTLAKRRTGVYVGVINH D--YERLILADRNSINAFSGLGRS TSIAA 75PKS506_STREP ISPREALAMDPQQRVVLETSWEA LEQAGIVPGTLRGSDTGVFMGAFS D---GYGLG--T--DLGGFGATGTQ TSVLSSALIA_PENGRI ISPKEAEQMDPQQRVSLEVASEA LEDAGIPAKSLSGSDTAVFWGVNS DD--YSKLVLEDLPNVEAWMGIGTA YCGVP
PKS-3_SUBDO GRISYRLGLQGPAVAVDTACSSSL VAIHQACQALRLGECDLALAGGA NVLLAPATMITFSNANMLAPDGKCK TFDAA 154PKS-5_SUBDO NRISYCFNLVGPSVTIDTACSSSL TAIDSACRALASQTVDYAFAGGA NAILSPESYIEFSQAAMLSKTGQCR AFDEK 152PKS506_STREP GRLSYFYGLEGPAVTVDTACSSSL VALHQAGQSLRTGECSLALVGGV TVMASPGGFVEFSQQRGLAPDGRCK AFAEASALIA_PENGRI NRISYHLNLMGPSTAVDAACASSL VAIHHGVQAIRLGESKVAIVGGVN ALCGPGLTRVLDKAGAISSDGSCK SFDDD ~~~~~~~ Cys~~ ß-ketoacyl synthas es active sitePKS-3_SUBDO ADGYVRGEGCGVIVIKRLEDAIS DGDRIRAVIRGSAINQDGASGGLT VPNGVAQQRVIADAIARADLEPSDV GYLEA 231PKS-5_SUBDO ADGFVRAEGGGLILLKRLSDALVD NDKIYASIIATSINQDGKTAGIM APSLDSQQAMMRDALNRCGICALDI GYVEA 229PKS506_STREP ADGTAFAEGSGVLVVERLSDAERH GHRVLAVVRGSAVNQDGASNGLSA PNGPSQERVIRQALANAGLQPSDV DAIEASALIA_PENGRI AHGYARGEGAGALVLKSLHRALLD HDNVLAVIKGSAVCQDGKTNGIM APNSVAQQLAANNALSAANIDPHTV RYVEA
ENVIRONMENT
TAXONOMY
speciesdiversity
ECOLOGYchemical ecology
GENETICSMOLECULAR
BIODIVERSITY(synthesis)
interspeciesdiversity
Survival mechanisms
Molecular cloning: success
S. raphanus
L. baicalensis
First decision: cloning
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HO
OHH
In vitro culture
Avarol
S. raphanus
L. baicalensis
10
extracellularspace
AF
plasma membrane
intracellularspace
Porifera
Continous epithelial surface Folding of pinacoderm and choanoderm
outside-in signaling: SELECTIVE GENE EXPRESSION
FN3
collagen
spicules
integrin
cell-matrix adhesion: TISSUE / SKELETON FORMATION
RPTP
cell proliferation and differentiation: GROWTH
EGF
soluble factors: MORPHOGENS
mucin
Wnt / Frizzled
RTK
secreted molecules: AXIS FORMATION
Forkhead
homeodomain protein
transcription factors: AXIS FORMATION
galectin
AR SRCR-SCR protein
putative AF
selectin: 86 kDa
36 kDa
cell-cell adhesion: CELL ASSOCIATION
galectin
Adhesion/signal transductionsystem
Sponges: PoriferaMonophyletic with all other Metazoa
Sponges: PoriferaMonophyletic with all other Metazoa
Urmetazoa
Müller (2006) The stem cell concept in sponges: metazoan traits. Seminars in Cell & Develop Biol 17: 481
11
Uriz - Gamulin
1899:DeLage Porifera = Mesozoa
Monophyly of Metazoa, Porifera excluded1988: Field
1787: Pallas Porifera = Thierpflanzen
1994: Müller-Gamulin Monophyly of Metazoa, Porifera included
1998: Borchiellini Monophyly of Metazoa confirmed
1999: Wiens M. et al. Poriferan 2-5A OA synthetase identified
1994: Gamulin-Müller Poriferan galectins identified
2000: Wiens M. et al. Poriferan apoptotic molecules
Sponges our ancestorsSponges: Porifera
Monophyletic with all other Metazoa
12
P. Westbroek – Y. LeGal
Nature as model
Bio-mineralization:Deep Sea
Xiaohong WANGNational Research Center for Geoanalysis, 26
Baiwanzhuang Dajie, CHN-100037 Beijing, P.R. CHINAE-mail: [email protected]
13
Enzymatic Biosilica
deposition
Biomineralization(biologically induced):
Deep Sea
Wang, Müller (2009) Marine biominerals: perspectives and challenges for polymetallic nodules and crusts. Trends Biotechnol 27: 375
China
Germany
USA
Nano-technologyChange of paradigm in biomaterial research
14
ChinaGermany
USA
Nano-biotechnologyChange of paradigm in biomaterial research
Nano-biotechnology
China
Germany
USA
Change of paradigm in biomaterial research
1999: Discovery of silicatein
2002: All patents in Mainz
(BIOTECmarin)
15
Change of paradigm
Synthesis of urea (Friedrich Wöhler, 1828)
Wöhler synthesis: conversion of ammonium cyanate (inorganic molecule) into urea(organic molecule - biochemistry)
For the first time an organic compound (urea) known to be produced only by biological organisms was produced in the laboratory, using chemical methods, from inorganic reactants.
Silicatein-catalyzed enzymatic synthesis of silica
Inorganic precursor Biosilica
For the first time an inorganic material (silica – glass) knownto be produced by an inorganic substrate synthesized by enzymes (silicateins) which are only by biological organisms.
Silicatein
Krasko A, Gamulin V, Seack J, Steffen R, Schröder HC, Müller WEG (1997) Cathepsin, a major protease of the marine sponge Geodia cydonium: Purification of the enzyme and molecular cloning of cDNA. Molec. Marine Biol. & Biotechnol. 6: 296-307
Cha JN, Shimizu K, Zhou Y, Christianssen SC, Chmelka BF, Stucky GD, Morse DE (1999) Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proc Natl AcadSci USA 96: 361-365
Biomineralization(biologically controlled):
Silicatein
16
Schematic outline of the effect of silica-based components (Na-
silicate, TEOS,nanoparticles) on the
expression of three genes [amelogenin, ameloblastin and
enamelin] in ameloblasts
New biomaterials
Bio-inspired morphogenesis:Nature: application
New Biomaterials:Osteoporosis
>40 >50 >60
17
New Biomaterials:Osteoporosis
Ossification: osteoclasts - osteoblasts
New Biomaterials:Osteoinductivity
Schematic outline of the coating process Schematic outline of the coating process of Caof Ca--P substratesP substrates
18
Encapsulation of bacteria in a silicashell: Fabrication of Nanofactories
Silicate coat
No effect on viability – growth
W.E.G. Müller, S. Engel, X. Wang, S.E. Wolf, W. Tremel, N.L. Thakur, A. Krasko, M. Divekar, H.C. Schröder (2008) Biomaterials 29, 771-779
Silicatein coating
New material
Bio-inspired morphogenesis:Nature: application
Müller, Engel, Wang, Tremel, Schröder (2008) Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-α gene. Biomaterials 29: 771