New pathways towards personalized medicine

Proteomics: the Search for Solutions For Neglected Diseases

Juan J. Calvete. Instituto de Biomedicina de Valencia, CSIC

Neglected diseases: an introduction

According to the World Health Organization (WHO), http://www.afro.who.int)¹, the Neglected Tropical Diseases, or NTDs, are a group of severe, chronic and disabling diseases which still persist, due in part to extreme poverty conditions, among the neglected populations of the 100 poorest countries of Africa, Asia and Latin America^2,3. The list of NTD recognized by the WHO include: helminthiasis (ascariasis, trichuriasis, necatoriasis, ancylostomiasis); schistosomiasis, dengue fever, lymphatic filariasis, trachoma, visceral leishmaniasis, African trypanosomiasis (sleeping sickness), American trypanosomiasis (Chagas disease), leprosy, dracunculiasis, Buruli ulcer, zoonotic parasitic diseases (cysticercosis, echinococcosis, fascioliasis, onchocerciasis); rabies; yaws; elephantiasis, strongyloidiasis and, since 2009, snakebite poisoning⁴.

About one sixth of the world’s population is affected by at least one NTD, and often by several of them; NTDs cause more than 534,000 deaths per year worldwide. The term “neglected” refers to the fact that many of these diseases could be prevented or eradicated by health programs combining political will and corporate philanthropy; assuming these programs were provided with funds adequate enough for researching, developing and distributing the drugs needed^2,5,6. More often than not, the diagnosing and treatment of neglected diseases require simple tests and low cost drugs; however, with unfortunate frequency, people living in remote, difficult-to-access areas die before the disease can even be diagnosed. The reason the NTD-affected populations are neglected by politicians and healthcare services is that these diseases affect impoverished populations who have little political leverage; and also, because these diseases do not represent an immediate danger for developed countries. Therefore, the neglect of these diseases condemns patients and their families to a vicious cycle of a loss of quality of life and productivity, which in turn leads to further poverty and marginality.

With the Millennium Declaration adopted on September 8, 2000, the United Nations asserted its determination to eradicate extreme poverty, hunger, and infectious diseases of great social and economic impact such as AIDS and malaria (http://www.un.org/millennium/declaration/ares552e.htm). Only five years later, the WHO and the CDC (Center for Disease Control and Prevention) defined NTDs as a great burden to the health and economic development of tropical and sub tropical low income countries^7,8. There are some clear examples of NTDs which can be controlled, or even eradicated, by using already existing, secure and efficient drugs. However, these cannot always be afforded by NTD-affected countries. For example, the elimination of trachoma in Morocco, Oman, and Mexico; the suppression of lymphatic filariasis in China, Thailand, Sri Lanka, Suriname, Solomon Islands, Trinidad and Tobago, Egypt, Costa Rica and Korea; or the elimination of onchocerciasis in ten west African countries, a success partly achieved thanks to drugs donated by the pharmaceutical industry^9,10. Despite these successes, it is worth noting that, although NTDs affect more than 1000 million people worldwide, less than 1% of those drugs registered between 1975 and 1999 were aimed at treating neglected tropical diseases.

Venomics and the Global Snakebite initiative

Snakebite envenoming is yet another example of a neglected pathology whose treatment, the administration of an anti-ophidic serum, has been known for more than 120 years¹¹. However, the insufficient production and distribution of efficient antisera, cheap enough to be afforded by the NTD-affected countries, causes more than 125,000 deaths each year and a much larger number of victims suffering from permanent physical or emotional disabilities^12,13. The lack of financial incentives for corporations to produce these serums, the shrinking of global markets and the weak leadership of public health organizations (both national and international) have left ignored the problem and its solution^14,15. Paradoxically, the venoms of the 725 or so species of poisonous snakes (or that of the 700 species of conus, 1,500 species of scorpions, 37,000 species of spiders, etc.) constitute a true pharmacological cornucopia, originated and refined by the process of natural selection during eons of biological evolution, a pharmacological cornucopia which creates an ocean of possibilities for the pharmaceutical industry¹⁶.

The implementation of “omics” high throughput ’omic’ technologies (genomics, transcriptomics, proteomics, metabolomics) to the study of venoms has led to the birth of a new discipline within toxinology, termed venomics¹⁷. Simultaneously, a group of researchers coming from the toxinology community, has launched, with the support of the International Society on Toxinology (IST, http://www.toxinology.org), an international initiative called the GSI, or Global Snakebite Initiative, http://www.snakebiteinitiative.org). Its main aim is the mobilization and coordination of the resources, capabilities, know-how and synergies of research, of clinical and technological development scientists, coming from the fields of venomics and toxinology, in order to search for solutions to snakebite envenoming^{13,18, 19}.

Achieving this aim requires close collaboration between NTD-affected countries and developed countries: the success of the GSI depends on developing a high level of coordination between those who know the real situation in the field and the problems of treating the disease, and those who have access to the “omics” technologies needed to study the molecular basis of the pathology and to identify targets for therapeutic intervention. It is noted that a similar initiative has managed to significantly reduce the incidence of Chagas disease in Latin America²⁰.

Proteomics versus Ophidism

Proteomics, that is, the large scale study of the protein complement of biological systems, has revolutionized the research of the structural and functional organization of living organisms. The application of proteomic techniques to the study of neglected diseases is experiencing an exponential growth, as evidenced by the large number of articles being published in specialized publications such as PloS Neglected Tropical Diseases (http://www.plosntds.org) or the Journal of Proteomics (http://www.journals.elsevier.com/journal-of-proteomics)²¹. The contribution to the GSI from our laboratory at the Valencia Institute of Biomedicine (which belongs to the Consejo Superior de Investigaciones Científicas, CSIC), is the development of proteomics techniques designed to elucidate the protein composition of snake venoms (“snake venomics”)²² and to analyze the neutralization effectiveness of commercial or experimental anti-venoms towards the toxins of homologous (i.e., included in the immunization mix) and heterologous (not included in the production of the antiserum as immunogens) venoms (“antivenomics”²³).

A detailed knowledge of the venom’s proteome and its pathological effects are essential prerequisites towards identifying the toxins causing the physiopathology of envenoming symptoms. Proteomic studies have demonstrated that the venoms of the snakes of the Viperidae genus (for example, European, African and Asiatic vipers) and Crotalinae genus (for instance, Asiatic Crotalus or American rattlesnakes) are composed of the combination of several types of toxins belonging only to a handful of protein families. The complexity of every specific type of venom depends on the type and relative abundance of the toxins expressed by it. Moreover, venomic research has evidenced the existence of phenotypic variability (geographical and ontogenetic) intra- and inter specific variability in the venoms’ composition. The identification of intra specific variations in the venoms of medically relevant species is very important for the rationalization of divergent symptoms, as well as for the selection of the best venom mix to create the corresponding antidote. A notorious case of intra-specific variation are the venoms of the Crotalus genus, that diverge in regards to the expression of toxins with neurotoxic, coagulant and hemorrhagic activity. The proteomic study of the venom of the American Crotalus showed that its neurotoxicity is a trophic paedomorphic trait evolved (i.e. retention of juvenile characteristics in the adult) in South American populations (as well as in some North American populations) during its evolutionary dispersion from the origin of the genus in Sierra Madre, in Mexico²⁴.

Aside from the possible ecological and taxonomical consequences, understanding the evolutionary trends of venoms by means of comparative venomics studies, as well as through the identification of converging and diverging characteristics among the different clades of the phylogenetic tree of ophidians, gives us clues for the elaboration of polyspecific antidotes. Thus, the experimental antidote for the neurotoxic venom of C. tigris also neutralizes the toxic activity of the paedomorphic venoms of North and South American species scattered across the evolutionary tree of Crotalus (Fig. 1).

Figure 1. Sketch of the phylogenetic tree of the Crotalus and Sistrurus genera highlighting the distribution of species expressing neurotoxic venoms that can be neutralized by antivenom serum generated against the venom of C. Tigris. It also shows the geographical distribution and pictures of C. tigris, C.d. terrificus and S.c. tergeminus.

On the other hand, the identification of converging venomics patterns between diverging species and the investigation of their degree of cross-immunogenicity may enlarge the range of therapeutic applications of existing antidotes. Thus, although the venoms of Central America snakes of the Bothriechis genus have very different compositions (Figure 2), the results of an antivenomics study have demonstrated that the anti-C. tigris antidote can also neutralize the neurotoxic effects of B. nigroviridis venom. The rationale of this observation is that both venoms contain high amounts of orthologous proteins of the crotoxin neurotoxin (i.e. the Mojave toxin).

Figure 2. Cladogenesis of the Bothriechis genus highlighting the enormous diversity of the protein composition of the venoms of three species studied through venomics techniques. The neurotoxic action of the B. nigroviridis venom (associated to the crotonix-like toxin PLA₂) can be neutralized by a antivenom generated against the venom of C. tigris (Figure 1.)

Hunters or Gatherers?

Physicist Freeman Dyson is reported to have remarked that scientific revolutions are more often driven by technological advances than by new concepts. “Omics” high throughput technologies have revolutionized the panorama of biological research, to the point that many laboratories using that technology have switched from the classical research paradigm based on making a priori hypotheses towards an hypothesis-free approach based on the a posteriori interpretation of huge amounts of data. Sydney Brenner called “hunters” to the former, and “gatherers” to the later²⁵. The more than 1,000 million NTD-affected people deserve greater attention from the scientific community; their access to the drugs needed to fight the effects of neglected diseases has become a human rights and ethical issue²⁶. However, none of the “omics” approaches explained above are appropriate for the search of proteomics-based solutions for NTDs. A new paradigm must be adopted, one that must be based on an in-depth knowledge of the biological system, a knowledge that only the hunters armed with the gatherer’s tools can achieve. However, they can only achieve it using the gatherers’ tools.

Cited Sources:
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[24] Calvete JJ, Sanz L, Cid P, de la Torre P, Flores-Díaz M, Dos Santos MC, Borges A, Bremo A, Angulo Y, Lomonte B, Alape-Girón A, Gutiérrez JM. Snake venomics of the Central American rattlesnake Crotalus simus and the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America. J. Proteome Res. 2010; 9:528-544.
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Other links of interest
http://apps.who.int/bloodproducts/snakeantivenoms/database
This WHO website includes information about the geographic distributions of medically relevant snakes, as well as a list of antivenoms and the species they neutralize.

http://www.reptile-database.org
This website contains information on all known snake species, including links to other sites of interest



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