Investigating the trypanosomatid lysosome and its role in lipid catabolism.

This project will lead to important new fundamental knowledge, allowing the development of lead compounds as novel therapeutics against the parasitic protozoa Trypanosoma brucei and potentially Trypanosoma cruzi, and related Leishmania species. These organisms are the causative agents of the neglected diseases; African sleeping sickness in sub-Saharan Africa (T. brucei) and Chagas disease in Central and South America (T. cruzi), "Old World" cutaneous leishmaniasis and "New World" visceral leishmaniasis (kala-azar) in East Africa, South America and Southeast Asia (various Leishmania species). Collectively these parasitic diseases affect millions of people and represent a huge percentage of the world's disease burden. Current drug treatments are woefully inadequate and there is an urgent need to identify novel therapeutic targets and to develop effective lead compounds.

Our research group has been working on lipid metabolism in these parasites, and it is clear that they have to scavenge many of the components required for them to make new membranes and divide and multiply. This has prompted us to try and understand better the role played by the parasites lysosome, which is equivalent to its stomach. This is where material that the parasite has to scavenge from the mammalian hosts bloodstream, is broken down into build blocks so that the parasite can make new membranes and cells so that it can divide and multiple.

Recently we have used methodology that has allowed us to purify some of these lysosomes away from other parts of the parasite, to specifically investigate what is inside them. Using state of the art techniques we are starting to identify a variety of proteins/enzymes that are able to break down host proteins and lipids. This novel information could be used by us and others, to find new drugs to inhibit these activities, thus starving the parasite of essential nutrients. This would be analogous to sailors who did not have enough fresh fruit, hence vitamin C, would get sick and die of scurvy.

The research proposed here will use a multidisciplinary range of different techniques across molecular parasitology and biochemistry. Several different approaches will allow us to identify the protein and enzyme functions within the parasites lysosomes, and thus what the parasite can and cannot digest.
This will help to inform us as to what structural features of compounds/pro-drugs could be used to either specifically by activated into active drugs and / or cannot be cleared and hence prevent the lysosome from working properly.
We have already identified several potential lysosomal enzymes involved in host lipid catabolism and want to confirm them as being in the lysosome, and essential, both genetically and chemically. The latter will be done by studying the enzyme activity, and then undertaking compound screening programmes to identify active compounds that can be modified to produce potent, and specific lead compounds that will form the basis of novel therapeutics.

This new knowledge will also aid current drug therapies from being more effective, if novel combinational therapies use compounds could be use to make the parasites sick and weaken. This would make the trypanocidal drug more effective, so overall lowering required dose, lowering costs, reducing the likely hood of side effects and chances of drug resistance, all criteria that would benefit the people and animals affected by these disease.

This research will deliver novel and invaluable knowledge that has huge potential towards forming new easy to make, affordable, easy to administer drugs in the fight against African sleeping sickness, as well as other closely related protozoan transmitted diseases.

This proposal follows up our purification of intact lysosomes from procyclic T.brucei. This study will advance our understanding of the parasites lysosome and the functions within it that are required for its survival. This multidisciplinary proposal will address the following:
1)Identify novel lysosomal proteins; initially focussing on the procyclic proteome, by undertaking a large-scale preparation. This will be followed up byin vitro preparations with bloodstream form, prior to using rodents to obtain larger amounts of material. Cellular localisation of some hits will be undertaken for validation purposes.
2)Study the enzyme activities associated with the lysosome; this will be done directly and indirectly. Directly, using tagged (fluorescently, radiolabelled, stable isotope labelled) probes (proteins or lipids) to investigate their catabolism, utilizing appropriate methodologies.
3)Validate novel drug targets associated with lysosomal lipid catabolism; we have identified a putative lysosomal proteins acidic sphingomyelinase, acid phosphatase and phosphodiesterase, that will be genetically validated. Localisation will be verified prior to recombinant expression, assays and screening efforts.
4)Undertake lipidomic / metabolomic studies of the lysosome; fractionation of the lysosomes obtained from under varying conditions, will allow lipidomic and metabolomics analyses
5)Identify new chemical entities that can interfere with lysosomal lipid catabolism; knowledge from this study, will highlight chemical entities (non-natural lipids) that cannot be processed by the lysosomal, thus poisoning the lysosomal activities.
Collectively this proposal will allow us to open a novel avenue of chemotherapeutic approaches against T. brucei and potentially other pathogens that rely heavily upon scavenging lipids and other essential metabolites from its mammalian hosts. We believe this work has real potential to impact on anti-parasitic drug discovery in the medium term.

Without overstating ourselves, we truly believe that this research will facilitate the identification of the next generation of useful drug targets and drugs that specifically target lysosomal enzymes in the fight against HAT and potentially other protozoan disease.
Impact beneficiaries from this research include the following:

1. The international scientific community and the public
The field in general will benefit from a plethora of new information on this poorly studied organelle in three distinct ways. Firstly, by ascertaining a lysosomal proteome. This new information will help us and others understand the enzymatic activities and proteins functions within this organelle, providing a rich seam of novel, potentially invaluable, drug targets. Secondly, the development of new methodologies that can be used to investigate metabolism and subsequent use of host nutrients by the parasites. Lastly, the genetic validation of drug targets and compound screening that will afford new chemical entities that can be used as lead compounds for further drug development against HAT, but also potential for a variety of other disease related research purposes.

2. Those involved in trypanosomatid drug research
Researchers in this area will benefit directly from new information on the mechanisms on host nutrient catabolism and usage by trypanosomatids. The data acquired during this research will have a crucial role in highlighting new research directions and priorities. The information obtained by this project could also become an integral part of future drug development process so that drug metabolism and potential resistance mechanisms are better understood and potentially avoided before drugs are licensed.

3. Those designing chemotherapeutic regimes.
The impact of our findings could have wide reaching implications for various trypanosomatid related diseases. A more complete understanding of how the parasites lysosome works will have important implications for drug discovery programmes and downstream treatment strategies for this and related neglected diseases caused by protozoa. This will allow policymakers to make informed decisions on which drugs to use and in which combinations to maximize their effect and limit undesirable factors.
The impact of our findings could have wide reaching implications for all three trypanosomatid diseases.

4. Patients infected with Trypanosomatid diseases
One product of this research project will be the considerable progress in the understanding of potential metabolic fates of novel drug treatments and their impact on novel combinational therapeutic regimes to be postulated against all three trypanosomatid diseases. These studies will aid in minimising the use of potentially toxic drugs and reduce the potential of refractory to treatment. This will have a huge impact on health and well-being as well as a positive social and economic impact on those people who need it the most.

5. The UK biomedical field
HAT, Chagas disease and leishmaniasis are some of the most "Neglected Diseases" of modern times. This project helps to strengthen the UK's presence in an internationally important biomedical research field and contributes to maintaining national expertise in this area. This is of additional relevance, given the steadily increasing number of cases of these tropical diseases now being detected within the UK due to international travel, as well as global warming expanding the range of the infected insects spreading the disease.

6. UK scientific links with countries of the sub-tropics
Many countries around the world in the sub-tropical region are undergoing rapid economic development. There has been a major transformation in their scientific infrastructure and it is hoped that the research in this proposal will initiate further collaborations and training links with scientific teams in several countries in South America, Central Africa and the southern half of Asia, to mutual benefit.