NTD Highlight Notice: Defining and leveraging the mechanism of action of suramin for treatment of trypanosomiasis.

Trypanosomatids are highly divergent protozoa; many, including the subject of this application, Trypanosoma brucei, are parasitic to humans and their livestock, while other species have huge impact on plants and the biosphere in general. Due to great evolutionary distance from humans, much of their biology is distinct from ours, and frequently cited as a potential opportunity for therapeutic intervention. Drug therapies are limited, with most being highly toxic and with emerging resistance. Due to complex surface architecture, vaccination is an unlikely therapeutic option. Of the available drugs, suramin, first introduced some 70 years ago, remains a drug of choice for early stage disease treatment. However, the mechanism(s) by which suramin is taken into the parasite or exerts trypanocidal activity have remained unknown.
T. brucei survives for long periods within many of its mammalian hosts, facilitated by antigenic variation. A single variant surface glycoprotein (VSG) is expressed to extremely high density on the parasite surface. Periodic switching of this coat serves to prevent immunological elimination of the entire population. A second aspect of the immune evasion system is highly efficient endosomal trafficking, responsible for removing surface-bound immunoglobulin. Further, several abundant non-variant surface proteins are also known; these 'invariant' surface glycoproteins (ISGs) are also internalized extremely efficiently. Two of the ISG families, ISG65 and ISG75 are turned over comparatively rapidly, via a ubiquitin-dependent mechanism. Endocytosis then may represent an attractive means to deliver compounds to the parasite interior, if a mechanism were available to target trypanocidal agents specifically to the trypanosome.
Most recently, we performed a genome wide screen using RNA interference to identify the gene products that are involved in the sensitization of trypanosomes to suramin. Many of these are involved in endocytosis, including the ISG75 family, but a substantial number have no known function. This application seeks to explore these proteins in more detail, and also to uncover additional factors that may play roles in suramin uptake and trypanocidal action. We shall examine if ISG75 represents the true receptor for suramin, link additional gene products with endocytosis, and explore the possibility of exploiting ISG75 as a means to specifically deliver trypanocidal compounds to the interior of the trypanosome, for therapeutic gain.
The program of work will further improve our understanding of the mode of action of suramin, delineating pathways of interaction with the parasite in even greater detail, and offering more precisely defined therapeutic insights and targets. Further, the program will explore a potentially novel approach to delivery of trypanocidal compounds emerging from this work, which may have broad applicability.

Treatment of protozoan diseases is difficult, partly due to limited local resources, resulting in a very sparse portfolio of drugs being available for some highly important diseases. African trypanosomiasis is invariably fatal unless treated; few are available, all of which have toxic side effects. Several have difficult and expensive administration regimens and resistance is highly prevalent. Further, our understanding of the modes of action of most drugs is limited. Suramin, introduced in the 1920s, represents one of the better compounds available, but is only active against early stage disease. Nevertheless, improved understanding of how suramin interacts with trypanosomes is of high value as this will uncover further potential points for therapeutic intervention. A recent genomic screen implicated a cohort of gene products in suramin sensitivity, including the surface protein ISG75, many endocytic proteins and a range of hypotheticals.
This proposal emerges from multiple ongoing investigations in our laboratories, including genome-wide screening, intracellular trafficking of surface proteins, specifically ISG75, and structural biology of surface proteins. We propose to determine if ISG75 is a bona fide suramin receptor and investigate the roles of specific ubiquitin hydrolases in ISG75 trafficking and suramin sensitivity; significantly, ubiquitylation is required for internalisation of ISG75. Further, to determine if the hypothetical proteins identified by us as part of the suramin sensitivity system are involved in endocytosis/ISG75 metabolism and identify the gene products involved in suramin toxicity in insect stages of the parasite and compare with the mammalian stage. The latter two aspects are targeted at determining if there are ISG75-independent routes (ISG75 is not expressed in the insect stage). The final aim is to investigate the potential of ISG75 as a delivery system for therapeutics, using specific antibodies as targeting mechanisms.

As a basic biology application, clearly most of the impact here is to individuals in directly related fields, and impact to the more general public is limited and rather non-specific by nature. However, clearly the advancement of understanding of drug action has a high public interest - the paper describing some of the work underpinning this application was published in Nature, featured in press releases and podcasts, attesting to broad impact from the work.
The RA will certainly benefit hugely. The application is highly cross discipline in nature, and will expose the RA to a great range of methods and collaborative opportunities. Specifically, analysis using the RNAi-based RITseq, imaging and biochemical approaches will provide opportunities to learn specific and advanced analytical methodology. Imaging and ultrastructural work will facilitate the exposure to these methods. As the project will encompass expertise from several laboratories, this will also provide great networking potential. The work will be presented at several international meetings, encompassing drug development, membrane trafficking and parasitology, again providing opportunity for networking, advancement and considerable broadening of expertise.
Most directly, research communities working with trypanosomes and their close relatives will see a major empowerment, and which includes the direct experimental community, parasitologists/trypanosomatid cell biologists working at the fundamental biology level, plus those working on direct impact of these organisms on health and agriculture. These individuals and their research goals will be aided in formulating improved hypotheses specifically targeting essential systems for study or therapeutic potential.
Next are researchers with interests in wider aspects of biology. Specifically here we consider the general protist and evolutionary biology communities. The analysis of drug mechanisms in trypanosomes is clearly of general impact, and the detailed mechanistic and novel methodological aspects of the work we seek to perform will broaden this impact. As the UK has a particularly large parasitology and trypanosome research community this is of specific benefit to the domestic scientific community.
Due to the broad geographical range of trypanosomes, beneficiaries will also include those with interests in ecology, environmental monitoring and human impact plus potentially climate change and agricultural management.
Further, individuals seeking to develop therapeutics will benefit from this work in the medium term - it is hoped that we will continue to refine our understanding of how the trypanosome interacts with suramin, and by identification of additional proteins, we anticipate that druggable opportunities will be identified and which can be exploited.
Finally, in public understanding and education: Trypanosomes and tropical diseases are organisms most school children and the general public encounter at some level, either more formally or in the media. Advances in understanding the biology of these organisms may increase the appeal and enthusiasm for basic and applied bioscience to the next generation and general public.