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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: wmueller@uni-mainz.de

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

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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

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Memantine

Flupirtine

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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

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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

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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

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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

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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: wxh0408@hotmail.com

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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

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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)

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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

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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

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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

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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