Molluscs: From Chemo-ecological Study to Biotechnological Application

Subseries of Prog ress in Molecular and Subcellular B iolog y

Series Editor:

Progress in Molecular and Subcellular Biology

Ph. Jeanteur, Y. Kuchino, A.Macieira-Coelho, R. E. Rhoads

Series Editors: W.E.G. Müller (Managing Editor)

Werner E. G. Müller

43

Marine Molecular Biotechnology

Volumes Published in the Series

Progress in Molecular Subseries:

and Subcellular Biology Marine Molecular Biotechnology

Volume 27

Signaling Pathways for Translation:

Stress, Calcium, and Rapamycin

R.E. Rhoads (Ed.)

Volume 28

Small Stress Proteins

A.-P. Arrigo and W.E.G. Müller (Eds.)

Volume 29

Protein Degradation in Health and Disease

M. Reboud-Ravaux (Ed.)

Volume 30

Biology of Aging

Volume 31

Ph. Jeanteur (Ed.)

Volume 32

I. Kostovic (Ed.)

Volume 33

W.E.G. Müller (Ed.)

Volume 34

Invertebrate Cytokines and the Phylogeny

of Immunity

A. Beschin and W.E.G. Müller (Eds.)

Volume 35

Dynamics

Ph. Jeanteur (Ed.)

Volume 36

C. Alonso (Ed.)

Volume 38

Ph. Jeanteur (Ed.)

Developmental Biology

of Neoplastic Growth

A. Macieira-Coelho (Ed.)

Molecular Basis of Symbiosis

J. Overmann (Ed.)

Volume 37

Sponges (Porifera)

W.E.G. Müller (Ed.)

Volume 39

Echinodermata

V. Matranga (Ed.)

Volume 42

Antifouling Compounds

Regulation of Alternative Splicing

Guidance Cues in the Developing Brain

Silicon Biomineralization

Epigenetics and Chromatin

A. Macieira-Coelho

RNA Trafficking and Nuclear Structure

Viruses and Apoptosis

Volume 43

Molluscs

N. Fusetani and A.S. Clare (Eds.)

Volume 40

Volume 41

G. Cimino and M. Gavagnin (Eds.)

Guido Cimino Margherita Gavagnin (Eds.)

Molluscs

From Chemo-ecological Study to Biotechnological Application

With 105 Figures, 9 in Color, and 18 Tables

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© Springer Berlin Heidelberg 2006

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E-Mail:

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Professor Dr. Guido Cimino

Professor Dr. Margherita Gavagnin

Italy

E-Mail: [email protected]

ISBN-10 3-540-30879-2 Springer-Verlag Berlin Heidelberg New York

ISBN-13 978-3-540-30879-9

Library of Congress Control Number: 2005937052

Istituto di Chimica Biomolecolare

Typesetting: SPI Publisher Services, Pondicherry

ISSN 1611-6119

Consiglio Nazionale delle Ricerche

Via Campi Flegrei, 34

80078 Pozzuoli (Naples)

Italy

Istituto di Chimica Biomolecolare

Consiglio Nazionale delle Ricerche

Via Campi Flegrei, 34

80078 Pozzuoli (Naples)

Cover design: design & production GmbH, Heidelberg, Germany

Cover illustration: nudibranch Jorunna funebris by courtesy of Dr. E. Mollo

Preface

The first volume of “Marine Molecular Biotechnology” – a subseries of

“Progress in Molecular and Subcellular Biology” - selected a very stimula￾ting topic: “Sponges (Porifera)”. The book proves that these animals are

only apparently simple. All chapters discover new scenarios with implica￾tions for evolution, associated microbiology, biodiversity, sustainable

exploitation and, of course, good science. This success prompted the

editors to continue this series selecting other topics. Professors Müller and

Schröder suggested “Molluscs” and we were generously invited to join

them in this exciting adventure.

Analogously to sessile organisms, slow moving marine invertebrates are

apparently without defence against both attacks from predators and

infections from micro-organisms even though they can select the best

habitat for their success in survival. Molluscs, and in particular gastropods,

fall in this category. They are generally protected by the shell and,

sometimes, also by toxins. Surprisingly, the venomous compounds from

some shelled molluscs can aid people to overcome the terrible pains of

terminal diseases. An example are the venoms of some Conus molluscs

which possess analgesic properties fifty times as stronger than that of

morphine. Other molluscs, the opisthobranchs, are only partially protected

by the shell. They were successful in their survival by constructing a very

effective arsenal of chemical weapons either sequestered from the

organisms upon which they feed or biosynthesized by themselves.

During the 70’s, many outstanding scientists (Prof. J. Faulkner, Prof. W.

Fenical and Prof. P. Scheuer) attracted the attention of the scientific

community with their exciting pioneering studies on opisthobranchs. Since

then, many groups have worked on this topic. The studies have moved

slightly from chemical ecology, to advanced biochemistry and applied

biotechnology. Many intriguing molecules have been isolated from

molluscs and some of them are now in an advanced clinical phase. Three of

the five PharmaMar compounds, at present tested in human clinical trials,

were detected by studying marine molluscs.

The volume “Molluscs” offers to readers an almost complete coverage

of the most stimulating topics related to molluscs, with the contributions

of many authoritative scientists active in this field. Organisms from all

seas are treated with the exception of those recently reviewed from the

Mediterranean Sea.

An explicative guide could be useful to the reader to navigate through

the volume. After an ecological introduction in the first chapter (Avíla),

toxins from bivalves and prosobranchs are extensively discussed in the

following three chapters (Uemura, Fattorusso and Marì). Darias reports a

comprehensive overview of the bioactive molecules from pulmonate

gastropods. The subsequent chapters deal exhaustively with molluscs from

distinct geographical areas, i.e. Antarctica, South Africa and South America

VI Preface

(Davies-Coleman), Australia and New Zealand (Garson), India, China and

Egypt (Wahidullah), and Japan (Miyamoto). Some relevant specific topics

are reported by Kamiya (bioactive proteins), Matsunaga (trisoxazole

macrolides), and Proksch (alkaloids). The two following chapters describe

biosynthetic studies on molluscs from the West coast of North America

(Andersen) and from Mediterranean littorals (Fontana) and introduce one

of the most intriguing topics exhibited by opisthobranchs: the ability to

construct de novo their bioactive compounds. At present, outstanding

groups in the world are very active in the synthesis of molecules isolated

from molluscs. However, this interesting topic is only partially treated here.

The synthesis of peptides and depsipetides (Spinella) has been selected due

to the very promising antitumor activity of these molecules. Finally, some

potent anticancer agents in clinical trials are described in the last chapter

(Cuevas).

“Molluscs” is dedicated to Prof. Kenneth L. Rinehart, Prof. Guido

Sodano and Prof. Salvatore De Stefano.

The outstanding scientific activity of K.L. Rinehart is mentioned in

Fernàndez’s foreword.

Here, we want to remember that the first work (1979) of our group and

many other studies on opisthobranchs were carried out thanks to the

valuable contribution of our colleagues and friends Guido and Salvatore.

Istituto di Chimica Biomolecolare (CNR) – Pozzuoli (Naples)

Guido Cimino and Margherita Gavagnin

Prof. S. De Stefano and Prof. G. Sodano

help in the editing work of this book.

Acknowledgements. We are deeply grateful to Mr. Raffaele Turco for his precious

Life originated in the oceans and has evolved there over a much longer

time than on land, so the diversity of life in marine habitats is far greater

than its terrestrial counterpart. Oceans cover nearly 70% of earth’s

surface and provide more than 90% of habitats for the planet’s life forms.

The first living organisms appeared in the sea more than 3500 million

years ago and evolutionary development has equipped many marine

organisms with the appropriate mechanisms to survive in a hostile milieu

in terms of extreme temperatures, changes in salinity and pressure, as

well as overcoming the effects of mutation, or bacterial and viral

pathogens. The diversity in species is extraordinarily rich not only in

coral reefs but also in other almost undisturbed natural marine habitats.

Marine organisms have developed exquisitely complex biological

mechanisms showing cross-phylum activity with terrestrial biota. In

terms of evolution and biodiversity, the sea appears to be superior to the

terrestrial ecosystem and marine species comprise approximately half of

the total biodiversity, thus offering a vast source from which to discover

useful therapeutics.

Several marine organisms are sessile and soft bodied. The question

thus arises: how do these delicate-looking simple sea creatures protect

themselves from predators and pathogens in the marine environment?

While answering this interesting ecological question, researchers found

that marine organisms have chemical defensive weapons (secondary

metabolites) for their protection. Outstanding taxa that are extremely rich

in those bioactive secondary metabolites are the mollusks. Intensive

evolutionary pressure from competitors, that threaten by overgrowth,

poisoning, infection, or predation, has armed these organisms with an

arsenal of potent chemical defense agents. They have developed the

ability to synthesize such chemical weapons or to obtain them from

marine microorganisms. Those compounds help them to deter predators,

keep competitors at bay, or paralyze their prey.

Investigations in the field of chemical ecology have revealed that the

secondary metabolites not only play various roles in the metabolism of

the producer but also in their strategies in the given environment. The

diversity of secondary metabolites produced by marine organisms has

been highlighted in several reviews and now comprehensively in this

monograph. They range from derivatives of amino acids and nucleosides,

macrolides, porphyrins, terpenoids, to aliphatic cyclic peroxides and

sterols. There is ample evidence documenting the role of these

metabolites in chemical defense against predators and epibionts. The

studies on marine chemical ecology in mollusks cover three different

aspects. Firstly, the diversity of chemical compounds produced by

different organisms; secondly, the potential functions of these metabolites

in nature: and finally, the strategies for their use for human benefit.

Preface by the Series Editor

VIII Preface

It is the merit of one of the most efficient experts working in the field of

marine natural products, Prof. Guido Cimino (Napoli), to have called

together prominent colleagues working in the field of natural products

from mollusca to highlight and push forward research on bioactive

secondary compounds from these animals. Guido Cimino is a pioneer

who succeeded in establishing that various patterns in the evolution of

chemical defense exist, including detoxification, modification, and

sequestration of metabolites and the positioning of those in places where

they will effectively repel predators. I am sure that this monograph will be

a platform for future successful developments in this field.

University of Mainz

Werner E.G. Müller

It is an honour for me to accept Professor Guido Cimino’s invitation to

Müller.

Mankind has always been very dependent on the sea, but the discovery

of a new source of medicines in the organisms living in the oceans has

opened up an enormously interesting new frontier. We founded

PharmaMar in 1986 to explore this new frontier. Today, I am even more

convinced of the potential of marine organisms as a source of medicines,

since the company has five marine anticancer compounds undergoing

clinical trials, with more than 4000 cancer patients treated so far. It is

relevant in the context of this book that three of these molecules have

been isolated from molluscs or derived from those present in molluscs, to

which this volume is dedicated.

I would also like to express my recognition to the scientists working in

marine organic chemistry who contributed to the discovery of those

antitumour molecules, which are derived from molluscs that are in

clinical trials: Professor Paul J. Scheuer for the discovery of Kahalalide F

from the sacoglossa Elysia rufescens; Professor Kenneth L. Rinehart for

the discovery of Spisulosine (ES-285) from the lamellibranch Mactromeris

polynima; Professor George R. Pettit for the discovery of the first

Dolastatin from the anaspidea Dolabella auricularia; and Professor Guido

Cimino for the discovery of Jorumycin from the nudibranch Jorunna

funebris, from which the PM-104 (Zalypsis®) analog is derived. And, for

the treatment of chronic pain, the conotoxin Prialt, which was discovered

by Dr. Baldomero Oliveira and his colleagues from the neogastropoda

I shall also take this opportunity to say a few words about Ken

Rinehart, who passed away a few months ago. It goes without saying that

Ken Rinehart was one of the most productive scientists researching

marine organic chemistry, and a point of reference that we will all sorely

miss in the future. I regret that he did not live to see ecteinascidin–743

(ET-743), which was discovered by his group, commercialised for the

treatment of certain cancers, such as ovarian cancer or sarcomas. When

these new treatments become available, I hope in the near future, they

will represent a legacy from Ken to the scientific community

Ken Rinehart was for many years a member of the PharmaMar Board

of Directors. He was also the person who selected the name PharmaMar

for our company. Throughout the years, he served on many scientific

committees where strategic decisions were made, and participated in

Foreword 1

Biotechnology” series, edited by Professor Werner E. G.

Conus magus, also deserves consideration.

write a foreword to the volume “Molluscs” of the “Marine Molecular

X Foreword

several PharmaMar scuba diving expeditions. We very much enjoyed

having him so involved with our company. Ken will always be with us.

PharmaMar – Madrid

José Maria Fernàndez Sousa-Faro

Prof. K.L. Rinehart

Molluscs are the largest of all marine invertebrate groups, consisting of

gastropods, bivalves, scaphopods, cephalopods, aplacophorans,

monoplacophorans, and polyplacophorans, many of which have been

widely used as food by humans. On the other hand, shells of gastropods

and bivalves have been used for making tools and ornaments.

Molluscs have been overlooked as biotechnological resources, except for

Tyrian purple (or royal purple), a brilliant dye derived from gastropods of

the superfamily Muricacea used in the eastern Mediterranean and in

China. Perhaps it represents the earliest documented application of

marine biotechnology. However, recent progress in marine

biotechnological research has shown that molluscs are potential resources

for biomedical and other biomaterials as partly described in this book.

Gastropods and bivalves sequester a variety of chemicals from food

organisms; bivalves often accumulate toxins from phytoplanktons referred

to as harmful algae (HABs) and cause food poisoning not only in humans

but also in marine mammals, which pose serious problems to food safety

as well as to marine environments. Therefore, HABs and shellfish

poisonings are an important area in marine biotechnology.

Opisthobranchs are a group of gastropods that are lacking in the shell for

physical defence, and have instead developed chemical defences. They

sequester defensive substances such as toxins, antifeedants, and

allelochemicals from their food, e.g., seaweeds, sponges, coelenterates,

bryozoans, and tunicates, which results in significant regional variations in

their defensive substances. The recognition mechanism of defensive

chemicals by nudibranchs may be applicable to many areas, especially to

drug delivery systems. The chemical defence of opisthobranchs is a good

model for understanding chemically mediated interactions of marine

organisms. A variety of unusual peptides isolated from herbivorous

opisthobranchs are powerful anticancer agents; several of them are

currently under human clinical trials. These peptides are actually of algal

origin, mostly cyanobacteria (blue-green algae). Fortunately, most of

these peptides can be supplied by chemical synthesis, differing from the

case of most marine natural product drug candidates.

Defensive compounds are also synthesized by gastropods. Particularly

interesting are polygodial and polypropionates, the former of which is

synthesized from mevalonates by nudibranchs of the genus Dendrodoris.

It is a wonder of nature that this powerful antifeedant is used for the same

purpose by terrestrial plants. Polypropionates, which are a rare class of

marine natural products, are contained in both pulmonates and

sacoglossans, the latter of which contain active chloroplasts sequestered

from green algae. Again, mechanisms of this sort of symbiosis and

chemical recognition are interesting subjects.

Foreword 2

X I Foreword

Proteins and peptides of molluscs have not been well explored.As found

in many animal species, a variety of antimicrobial peptides (AMPs) and

proteins are reported from bivalves and gastropods. They are involved in

innate immunity and potential antimicrobial agents. Cone snails contain

numerous numbers of small peptides tabbed as conotoxins possessing

various pharmacological activities, most of which have enormous

therapeutic potential. In fact, •-conotoxin MVIIA, an N-type Ca2+ channel

blocker, has recently been approved as an analgesic in the USA.

Biopolymers such as glue proteins produced by bivalves, especially

mussels, have potential for biotechnological applications.

Cephalopods are unique among molluscs; they can swim fast and use

ink for defence. Perhaps this property prevents them from having

interesting chemicals for their defence. Biotechnological investigation of

cephalopods is very limited.

There is no doubt that molluscs are an important biotechnological

resource as briefly mentioned above. Obviously, we need to exploit them

more deeply from application-oriented viewpoints.

Hokkaido University

I

Nobuhiro Fusetani

Molluscan Natural Products as Biological Models: Chemical Ecology,

Histology and Laboratory Culture................................................................... 1

Conxita Avila

1 Introduction ........................................................................................... 1

2 Chemical Ecology of Molluscs ..............................................................5

3 Histology: from Tissues to Cell Location.............................................9

4 Laboratory Culture: Producing Bioactive Compounds.....................11

5 Conclusions .......................................................................................... 13

References........................................................................................................ 15

Shellfish Poisons............................................................................................... 25

Masaki Kita, Daisuke Uemura

1 Introduction ......................................................................................... 25

2 Pinnatoxins, Ca2+ Channel-Activating Polyether Toxins from the

Okinawan Bivalve Pinna muricata.....................................................26

2.1 Isolation and Structure of Pinnatoxin A............................................26

2.2 Structure of Pinnatoxins B and C .......................................................28

2.3 Biological Activity of Pinnatoxins......................................................29

2.4 Biogenesis and Synthesis of Pinnatoxins...........................................29

2.5 Symbioimine, a Potential Antiresorptive Drug.................................30

3 Pteriatoxins, Pinnatoxin Analogs from the Okinawan Bivalve

Pteria penguin – Nanomole-Order Structure Determination ......... 32

3.1 Isolation of Pteriatoxins ...................................................................... 32

3.2 Structure of Pteriatoxins ..................................................................... 33

3.3 Other Macrocyclic Iminium Toxins Related to Pinnatoxins ...........36

4 Turbotoxins, Diiodotyramine Derivatives from the Japanese

Gastropod Turbo marmorata .............................................................38

4.1 Isolation and Structure of Turbotoxins .............................................38

4.2 Structure–Activity Relationship .........................................................39

5 Pinnamine and Pinnaic Acids, Alkaloidal Marine Toxins from

Pinna muricata..................................................................................... 41

5.1 Pinnamine............................................................................................. 41

5.2 Pinnaic Acids: cPLA2 Inhibitors.........................................................42

5.3 Halichlorine: an Inhibitor of VCAM-1 Induction .............................43

5.4 Biogenesis of Pinnaic Acid..................................................................45

6 Conclusions ..........................................................................................46

References........................................................................................................46

Contents

Bivalve Molluscs as Vectors of Marine Biotoxins Involved in Seafood

Poisoning ........................................................................................................... 53

Patrizia Ciminiello, Ernesto Fattorusso

1 Introduction ......................................................................................... 53

2 Marine Biotoxins.................................................................................. 55

2.1 Paralytic Shellfish Poisoning............................................................... 57

2.1.1 Paralytic Shellfish Toxins.................................................................... 57

2.1.2 Clinical Symptoms of PSP ...................................................................58

2.2 Diarrhetic Shellfish Poisoning ............................................................59

2.2.1 Diarrhetic Shellfish Toxins .................................................................59

2.2.2 Clinical Symptoms of DSP ................................................................. 60

2.3

2.3.1 Pectenotoxins ...................................................................................... 60

2.3.2 Yessotoxins........................................................................................... 61

2.3.3 Azaspiracids .........................................................................................63

2.4 Neurotoxic Shellfish Poisoning ..........................................................63

2.4.1 Neurotoxic Shellfish Toxins................................................................64

2.4.2 Clinical Symptoms of NSP ..................................................................65

2.5 Amnesic Shellfish Poisoning.............................................................. 66

2.5.1 Amnesic Shellfish Toxins ................................................................... 66

2.5.2 Clinical Symptoms of ASP...................................................................67

2.6 Spirolides and Shellfish Syndrome Related to Dinoflagellates........67

3 DSP Toxins in Phytoplankton and Mussels from the

Northwestern Adriatic Sea ..................................................................68

3.1

3.2 LC-MS Method for Analysis of YTXs.................................................70

3.3 LC-MS Analysis of an Adriatic Strain of P. reticulatum...................72

4 Detection of Domoic Acid in Adriatic Shellfish by Hydrophilic

Interaction Liquid Chromatography–Mass Spectrometry .............. 73

5 Cytotoxins from Contaminated Adriatic Blue Mussels....................74

5.1 Oxazinins ..............................................................................................74

5.2 Chlorosulfolipids ................................................................................. 75

6 Conclusions ..........................................................................................76

References........................................................................................................77

Hyperhydroxylation: a New Strategy for Neuronal Targeting by

Venomous Marine Molluscs ...........................................................................83

Aldo Franco, Katarzyna Pisarewicz, Carolina Moller, David Mora,

Gregg B. Fields, Frank Marì

1 Introduction .........................................................................................83

2 Hydroxylation of α-Conotoxins .........................................................87

3 Hydroxylation of Mini-M Conotoxins...............................................89

VI Contents X

Toxins Found in Association with DSP Toxins................................ 60

New YTX Analogs Isolated from Adriatic Mussels ..................... 69 .....