Animals’ venoms to improve human health: a new approach with promising results
After 30 months, 120 poisonous species have been collected, 90 of which have been analysed by transcriptomics and 30 of them by proteomics. 20,000 more sequences are expected to be gained at the end of the project, the greatest database of the existing toxin sequences so far. The discovery of hundreds of peptides and proteins in the venom of only one poisonous species opens multiple opportunities for pharmacological research
Lisbon, June 24 - In nature there are many keys that could open the holy grail of drug discovery of the 21st century; specifically, one of these keys to optimize human health in the near future is expected to be found in the venom of animals.
And the fact is that the poisons constitutes one of the most promising sources for the creation of new pharmacological compounds for the treatment of human diseases, due to its functional activities, small size, low immunogenicity and stability. Furthermore, the development of strategies and powerful technological features, which facilitate chemical synthesis or recombinant production, has increased the impetus to study this resource. This interest is further justified if you consider that there are over 170,000 venomous animals and the animal poison brings in a collection of over 40 million compounds, of which only about 5,000 are known to this date and have been adequately studied.
Settling this lack of knowledge and exploiting the potentials for the human health using the study of animal venom is the challenge of Venomics project, a European initiative with 8 private and public partners, which has the primary purpose of exploring and exploiting the poisons as a resource to develop innovative medicines. Among its challenges is to answer questions of the researchers and healthcare organisations. Can animal toxins offer new possibilities to cure human diseases? Are there still animal toxins that are unidentified and are not studied? Is it possible to do this without having any prior knowledge of the analysed poison and with a minimal amount of it? Would new sequencing technologies help to identify new toxin candidates? Is it possible to do this more efficiently, cost effectively and faster than it has been done so far?
Today the first results of this project, which now reaches its halfway point and is funded by European funds (with a budget of €9.1 million of which €6 million are funded through the Seventh Framework Programme FP7 HEALTH FP7, 2011-2015) were presented in Lisbon. Leading companies from five European countries (Belgium, Denmark, France, Portugal and Spain) are participating in this initiative.
Halfway point of an ambitious and promising project
“It is the biggest project to date in this field in the whole world, exploring the ability of poisons to produce drugs at a scale that has never been produced before, with more than 200 investigated poisons" says Dr Nicolas Gilles, General Project Coordinator. The scientific team has travelled to different parts of the world (especially in French Guiana) to collect the samples, most of them had never been analysed and characterised before.
The project also includes a cutting-edge technology that will allow faster drug development with lower cost. The diseases that are included in this study are pain disorders, cardiovascular disease, cancer or diabetes.
Thirty months after starting the project, 120 poisonous species have been collected, of which 90 have been analysed by transcriptomics and 30 by proteomics. Of the top ten poisons for which final information is available, an average of 200 sequences per each species were identified. "We are hoping to obtain a bank of 20,000 sequences at the end of the Project which will represent the greatest database of toxin sequences that has been constructed so far", says Dr Nicolas Gilles, General Project Coordinator and Head of the Laboratory of Molecular Engineering of Proteins from Institute of Biology and Technology Saclay ECA (French Alternative Energies and Atomic Energy Commission, France), who notes that "the latest technical development that has occurred in this project is the beginning of the production of toxins."
According to this expert, “the project will have a major impact on public health, offering both the possibility of developing innovative drugs directed to target receptors and the new therapeutic targets and as new therapeutic ways for a set of medical needs that are not being covered currently”
Animal poison, the key to improve human health?
Poisons are mostly studied for their toxic effects, while their natural pharmacological properties represent a great source for the creation of new classes of drugs. At present, six toxins, or toxin-derived drugs are already on the market, which are suitable for chronic pain or regulate blood clotting.
As defined by Dr Frédéric Ducancel, Head of the Department of Pharmacology and Immunoassay from Institute of Biology and Technology of Saclay CEA (France), "animal venoms are complex cocktails which contain several hundreds of components, most them are proteins or peptides". Their interaction with enzymes, ion channels, receptors and other elements can result in direct or indirect effects on the integrity of the cells, the central nervous system and/or peripheral muscles or blood flow.
This gives them a special interest, mostly because it is assumed that the poisons are natural reservoirs that contain many bioactive molecules that have been selected and recruited for their secretion, structural stabilisation, functional plasticity and the ability of participation in molecular interactions. As noted by Dr Ducancel, "the incredible potential of poisons for drug discovery makes them particularly interesting for the pharmaceutical industry, which is in extreme need of innovating".
A lot of toxin targets are implicated in several human diseases, such as pain, cancer, neurodegenerative diseases, cardiovascular disease, diabetes, obesity or depression.
A breaking vision
VENOMICS comes up with an innovative way of approaching to the study of poisons, presenting a new change in conceptual point of view as a scientist.
On one hand, the poisons are not analysed as a toxic material but rather as a natural resource which consists of peptides that can have innovative pharmacological activities and may be useful in human healthcare. "Among 170,000 different poisonous species that contain 100 to 500 toxins, we estimate that there are 40 million of peptides; however, by now we only know about 5,000 of all of them." says Dr Nicolas Gilles, General Project Coordinator.
But in addition, the Venomics project offers an innovative scientific approach. Instead of relying on the classical bioassay-guided approaches (low yield for the identification of bioactive peptides, which requires large amounts of candidate poisons and focuses primarily on abundant compounds in the venom), VENOMICS suggests an entirely new paradigm that overcomes some past limitations.
“Omics technologies are applied to understand better the diversity of venom and generate peptide sequences; then it will proceed to in vitro mass production of compatible peptides detected by next generation sequencing", reports the General Project Coordinator of Venomics.
Venomics flow work is divided into four main steps:
- Study sources. The CEA (French Alternative Energies and Atomic Energy Commission) takes samples of big and small poisonous animals as they collect samples in French territories (like French Polynesia and Mayotte).
- Sequencing database. Proteomic analysis of the poisons is conducted by the University of Liege (Belgium), while transcriptome analysis of venom glands is performed in Sistemas Genómicos (Valencia, Spain). Through these assessments, the additional information is provided which is a combination of all of them (CEA, France), generating the list of toxins in each venom.
- Peptide bank. The toxins that are selected from the sequencing database are manufactured by recombinant expression (NZYTech-Portugal- and University of Marseille-France-) and peptide chemistry (CEA, France).
- Creation of the new drugs. The accumulated information in the peptide bank eventually leads to therapeutic targets related to unmet medical needs (Zealand Pharma, Denmark, Catholic University of Leuven, Belgium and CEA, France).
A new approach with new tools
Venomous animals have a huge arsenal of cross-linked peptides which are used for their defence and predation. Their enormous structural and pharmacological diversity makes them an excellent basis to identify innovative drugs.
In this regard, mass spectrometry and transcriptomic studies have demonstrated the presence of several hundreds of peptides and proteins in the venom of each animal species. Instead of relying on the classical approach, low performance for the identification of bioactive peptides, the European project FP7 VENOMICS proposed an entirely new paradigm to access the diversity of poison.
As indicated by Professor Edwin De Pauw, responsible of the Venomics Proteomics Project Area, "by combining proteomics and transcriptomics technologies we can generate reliable sequences of peptides." As added by this expert, Professor of Physical Chemistry and Head of the Mass Spectrometry Laboratory of the University of Liege (Belgium), "proteomics technology allows us to analyse even very small toxins (<5 kDa), which led us to determine over 150 toxins sequences for each venom. "
These analyses are completed by transcriptomic study that is conducted in the Spanish company, Sistemas Genómicos. Transcriptomics is the study of gene expression in a cell, tissue or organ, and is based on the sequencing and the analysis of RNA molecules which are the result of the gene expression in a specific condition.
Specifically, Sistemas Genómicos has developed de novo transcriptomics, an innovative methodology that enables identification and expression analysis of RNA molecules or transcripts of interest (such as toxins), without any prior knowledge about the organism. This new technology offers a great potential for the discovery of new RNA molecules, or transcripts.
"This powerful application allows the identification of unknown molecules that had not been explored before, opening up huge possibilities for the discovery of new drugs," says Rebecca Miñambres, Responsible of Transcriptomic Area of Venomics Project.
Moreover, with the incorporation of new technologies such as Next Generation Sequencing (NGS), millions of sequences that are subsequently assembled through bioinformatic processes can be obtained. This method facilitates, with a high resolution, the identification of transcripts (even those with very low expression).
In terms of sequencing, the challenge of this project was setting up all the protocols in the laboratory for these special samples, due to the low amounts of material that can be obtained from the venom gland of an animal as small as a wasp, an ant or a tiny spider. The "low input protocols" have been developed to adjust the sequencing process for very small amounts of RNA.
"We are now working with 50 to 200 nanograms of RNA, while at the beginning of the project at least 1,000 nanograms of RNA for sequencing were necessary, so we have reduced 5 to 20 times of the amount of primary required material, always regarding to the quality of the sample, "says Rebecca Miñambres, Head of the Projects Department in Sistemas Genómicos.
On the other hand, the bioinformatic assembly of the obtained sequences was another challenge. 12 GB of information from each sample is obtained, including billions of short sequences provided by NGS platforms.
With these short sequences an entire map of complete transcriptomes of the organisms is created, which enables the identification and quantification of all the genes that are expressed in the venom gland. "We need to reconstruct the complicated jigsaw with all of the pieces to obtain a set of genes that are expressed in the venom gland," says Dr Miñambres.
In some of these genes the toxins may exist and other genes may be related to many different cellular functions; besides, the transcripts, RNA molecules, of the above known toxins will be obtained and the other ones will be considered as novel toxins that were not fully characterised before.
Video of the Press Conference in Lisbon: