Drawing on his expertise in marine toxins and pharmacology, Professor Luis M Botana is conducting fascinating research into the mechanisms of neuroprotective compounds derived from the Earth’s oceans. Here, he discusses the importance of collaboration and the challenges he has overcome to date

Secrets in the sea

How has your professional background led to your interest in finding novel neuroprotective compounds to combat dementia?

I have been working with marine compounds for over two decades and am fascinated by their complex mechanisms of action and potential as drug leads. Over time, my colleagues and I observed that many of these molecular mechanisms appeared to have an effect on neurons, and so we began to explore whether they had any impact on neurodegenerative diseases such as Alzheimer’s. We found that their different modes of action have great potential for anti-neurodegenerative drugs.

Could you provide an overview of your experimental process and what you hope to accomplish through your research?

Our work is primarily conducted on the basis of invaluable collaborations with other scientists who are experts in the collection, isolation, identification and synthesis of many of the marine compounds that we use. These compounds, in pure form, are obtained from oceans in many different parts of the world and include deep sea samples, sponges, bacteria, dinoflagellates, fish and snails, among others. Once we have these compounds in our laboratories, we screen them to identify their mechanisms of action. We study those that show promising effects for potential therapeutic targets, mostly using transgenic animal lines that express characteristics of dementia from birth.

What are the most important neuroprotective compounds with which you work? To what extent do their mechanisms of action vary?

Initially, we focused on the marine compound yessotoxin. Even today, its mechanisms of action are not fully understood, although we think they can largely be attributed to the enzyme phosphodiesterase 4. This enzyme is linked to processes like apoptosis and necrosis and is extremely complicated to analyse. Interestingly, yessotoxin is not only a potential anti-Alzheimer’s drug lead, it also appears to have anticancer effects and applications in the treatment of allergies and diabetes.

Yessotoxin increases the expression of the genes responsible for the production of the low density lipoprotein receptor, hexokinase 1 and 2, fructose-2,6-biphosphate, fatty acid synthase and acetyl CoA carboxylase, all of which are related to the biochemistry of Alzheimer’s disease. It is important, although not well understood as yet, that there are biochemical links at the neuronal level between diabetes and Alzheimer’s disease. So far we have generated five different patents for the therapeutic use of yessotoxin but developments have been stalled as a consequence of their complex chemistry.

Have you faced any particular obstacles to your work? How have these been overcome?

The main challenge we face is the enormous cost involved in conducting this type of

research. Through some international projects, such as BAMMBO or PHARMASEA, we have been able to coordinate our screening efforts with the isolation work of other relevant groups in Europe, Japan, Chile, India and the US. This international dimension is essential, not only because it provides us with access to different geographical samples, but because it also enables us to access the expertise of other researchers and share the heavy financial burden.

Your research has demonstrated the potential for the use of secondary metabolites in drug development for neurodegenerative diseases. How do you foresee this study progressing?

The question itself poses an interesting philosophical debate. Are these compounds really secondary metabolites or are they in fact something else? Why do toxins only affect terrestrial animals and not those in the marine ecosystem? Why do some organisms expend so much energy and so many genetically redundant resources to produce compounds that do not appear to have a clear ecological function? If we could answer these questions we would be better placed to address other relevant aspects regarding the biosynthesis and targets of these compounds. For us, it is clear that the largest drug potential for the future resides in the sea, not only because of the vast number of unknown and potent compounds contained within the oceans but also because they have been far less explored than those on land.

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PROFESSOR LUIS M BOTANA

SPREAD OVER 70 PER CENT of the Earth’s surface, oceans contain a vast number of ecosystems and an estimated 50-80 per cent of all life on Earth. The diverse organisms found within have evolved to adapt to hostile environments, including extreme temperatures, high salinity and high pressure. To aid their survival, marine life forms produce a large number of extremely active compounds that serve a variety of biological purposes. Interestingly, recent years have witnessed concerted efforts to unlock the potential of these in the context of drug development.

Many of the toxic marine compounds under scrutiny represent particularly promising pharmacological tools in the treatment of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. This is important as longer life expectancies have led to the increased incidence of age-related neurological diseases, in turn constituting an enormous socioeconomic burden in developed countries throughout the world. With their incidence projected to continue growing exponentially over the coming decades, research that unveils potential therapeutic strategies is of the utmost importance.

A WORLD-LEADING GROUPBased in the Faculty of Veterinary Medicine in the Department of Pharmacology at the University of Santiago de Compostela, Professor Luis M Botana’s research group is leading the way in studies of marine toxins. Working in collaboration with a number of research institutions and scientists from all over the world, the group benefits from unfettered access to international experts in toxicology, pharmacology and therapeutics. Together, the researchers are exploring the action mechanisms of marine compounds and their potential for drug development, as well as the design of innovative detection methods.

The sheer variety of marine organisms – spanning bacteria, dinoflagellate, fungi, sponges, micro- and macroalgae – is immense, offering enormous potential as a source of new therapeutic compounds “Many marine compounds have neuro-active effects on terrestrial mammals, throwing up possibilities for modifying their structure in order to increase their beneficial effects and reduce

their toxic effects,” elaborates Botana. “In many cases, however, the chemistry of these compounds is extremely complex, meaning that they pose a major challenge to organic synthesis.” Compounds such as yessotoxin and ciguatoxin, for instance, are cases in point; they have large molecular weights and highly complicated mechanisms of action. Indeed, while drug companies are aware of the enormous potential of marine toxins, their complexity – in addition to international legal issues regarding the intellectual property of marine organisms – means that they have not been exploited as extensively as they deserve.

OVERCOMING OXIDATIVE STRESSOne of the most promising lines of research being pursued by Botana and his team is centred on the potential antioxidant and neuroprotective effects of marine compounds. Indeed, oxidative stress has long been associated with neuronal cell death in the ageing process, and with the development and progression of neurodegenerative diseases. Caused by an imbalance in the production of reactive oxygen species (ROS) and the body’s subsequent inability to detoxify these and repair associated damage, oxidative stress induces toxic effects through the production of free radicals that harm cells.

As the body ages, it becomes increasingly sensitive to the damaging effects of free radicals; neurons often bear the brunt of this as the brain consumes the vast majority of oxygen in the body: “The reason neurons are particularly affected is because their rate of metabolic activity is very high, meaning that the production of free radicals and their deleterious effects are also high,” Botana explains. “Additionally, neurons are rich in phospholipids and iron, which react with oxygen. For example, beta amyloid production in Alzheimer’s and alpha sinuclein production in Parkinson’s are both products of oxidative stress suffered by neurons.”

A particularly key study conducted by Botana and his team focused on the bioactive chemicals found in marine sponges and their

role in preventing oxidative stress. Together, the researchers investigated the impact of six diterpene derivatives isolated from Spongionella on mitochondrial function in the cortical neurons. They co-incubated the compounds with hydrogen peroxide for 12 hours in an attempt to establish whether or not they had a protective effect on cell death and the pathways that regulate production of antioxidant proteins. Revealingly, using their in vitro oxidative stress model, they were able to demonstrate that these compounds induce neuroprotective activity by combating oxidation.

Going forwards, while such results are encouraging, there is a need to keep investing in robust international collaborations and continue investigating the underlying mechanisms by which diverse, bioactive compounds mitigate the damaging effects of ROS. As Botana’s research demonstrates, marine toxins seem to be an extremely promising pharmacological tool for age-related diseases. Looking ahead, however, their potential extends far beyond treatments for neurodegenerative diseases; they could also be used as drugs for fungal diseases, allergies and diabetes. For now, Botana and his collaborators are planning to apply their ingenuity and expertise to uncovering the secrets of these complex and fascinating compounds.

Many toxic marine compounds under scrutiny represent particularly promising pharmacological tools in the treatment of neurodegenerative diseases such as Alzheimer’s and Parkinson’s

The Department of Pharmacology at the University of Santiago de Compostela, Spain, hosts a group of researchers committed to exploring the potential of a diverse range of marine organisms for use in the development of therapeutics for neurodegenerative diseases

Marine metabolites

Prorocentrum minimum. Gambierdiscus toxicus.

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COMPELLING COMPOUNDS

In addition to shining the spotlight on bioactive toxins in marine sponges, Botana and his colleagues have also explored the capacity of other ocean-derived compounds to fuel the development of neurodegenerative drugs.

Gambierol

As a potent neurotoxin from the family of marine polycyclic ether natural products, gambierol is known to target voltage-gated potassium channels in excitable membranes. In their study, Botana and his team, in collaboration with Professors Haruhiko Fuwa and Makoto Sasaki from Tohoku University, Japan – who designed and synthesised two truncated skeletal analogues of gambierol – analysed their effect on a mouse model of Alzheimer’s disease. Promisingly, their results demonstrated that pre-incubating the cells with the compounds led to the inhibition of potassium currents, the decrease of extra- and intracellular levels of amyloid beta and the reduction of hyperphosphorylated tau levels. Ultimately, this study highlights how gambierol and its synthetic analogues can advance knowledge about the function of potassium channels and the molecular mechanisms of amyloid beta metabolism.

Spirolides

Although spirolides are not currently used in routine monitoring assays, these marine toxins appear to target nicotinic receptors, and previous in vitro studies have flagged up their positive effects in the context of Alzheimer’s disease. Using a well-established Alzheimer’s mouse model, Botana and his colleagues identified neuroprotective effects following the administration of low intraperitoneal doses of spirolides.

Streptomyces

Streptomyces is a family of bacteria largely found in soil and decaying vegetation. Using their cutting-edge screening tools, Botana’s team examined seven natural products

from Streptomyces sources, demonstrating their ability to inhibit neuronal cytotoxicity and reduce the release of reactive oxygen species. These findings position Streptomyces metabolites as strong candidates for the development of new drugs.

Bromoalkaloids

In a recent study, Botana and his collaborators investigated the neuroprotective and antioxidant abilities of hymenialdisine and hymenin, two members of the bromoalkaloid compound family, obtained from Porifera. They used an oxidative stress cellular model to analyse several oxidation biomarkers. Their results demonstrated that the two compounds had strong neuroprotective abilities at low nanomolar concentrations.

Diterpenes

In recent work, they also investigated the effect of gracillins – diterpene compounds obtained from the sponge Spongionella – that show a promising effect to protect against oxidative stress and as anti-Alzheimer leads.

SPONGIONELLA SECONDARY METABOLITES PROTECT MITOCHONDRIAL FUNCTION IN CORTICAL NEURONS AGAINST OXIDATIVE STRESS

OBJECTIVES• To study the mechanism of action of novel

marine compounds sourced from dinoflagellates, sponges, bacteria, etc., to identify new potential therapeutic leads for neurodegenerative diseases

• To use the targets of those compounds that are toxic to humans to develop new methods of detection for the prevention of human intoxications

KEY COLLABORATORSProfessor Olivier Thomas, University of Nice, France • Professor Jordi Molgo, French National Centre for Scientific Research (CNRS) • Professor Vitor Vasconcelos, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Portugal • Professor Chris Elliott, Queen’s University Belfast, UK • Professor Marcel Jaspars, University of Aberdeen, UK • Professor Kiriakos Nicolaou, Scripps Research Institute, USA • Professor Takeshi Yasumoto, National Food Research Institute, Japan • Professor Haruhiko Fuwa; Professor Makoto Sasaki; Professor Masahiro Hirama, Tohoku University, Japan • Professor Patrick Murray, Limerick Institute of Technology, Ireland • Professor Daniel Romo, University of Texas, USA

FUNDINGEuropean Union Framework Programmes

Spanish Funding

CONTACTProfessor Luis M BotanaFull Professor of Pharmacology

University of Santiago de CompostelaSpain

T +34 982 822 233E luis.botana@usc.es

@luismbotanaLuis M Botana

PROFESSOR LUIS M BOTANA gained his undergraduate degree and PhD in Pharmacology from the University of Santiago de Compostela before taking up a

position as Fogarty Fellow at Johns Hopkins University, USA. Over the course of his career he has co-authored 250 peer-reviewed scientific papers, holds 25 patents and has edited 10 books.

INTELLIGENCE

Bands in low pressure separation of marine extracts.

Most of the research group at the Department of Pharmacology in Lugo, Spain.

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