NTD Highlight Notice: Melarsoprol-pentamidine cross-resistance in African trypanosomes

African trypanosomes are protozoan parasites that cause a range of important human and animal diseases. Human African Trypanosomaiasis (HAT) is typically fatal without drug treatment. Transmission is via the tsetse fly and parasites then circulate in the bloodstream and tissue fluids of their mammalian hosts. The public health situation has improved recently with increased monitoring and therapy averting more than 1.3 million DALYs (Disability-Adjusted Life Years) during 2000 and cases estimated at less than 70,000 in 2006. However, therapies suffer multiple problems including severe toxicity and increasing resistance, a major concern due to the absence of a vaccine or therapeutic alternatives. The only current HAT therapies in most cases are pentamidine, which is only suitable for the first stage of the disease prior to central nervous system involvement, and melarsoprol for advanced disease. Melarsoprol contains arsenic and causes a fatal reactive encephalopathy in around 5% of patients. This desperate situation is made worse by the current and increasing incidence of melarsoprol treatment failures; 20-30% in Uganda, the Democratic Republic of Congo, and the Sudan. This is due to the spread of drug resistance. In fact, the problem is one of multi-drug resistance to both pentamidine and melarsoprol. The applicant's group recently made a breakthrough in this area and linked a defect in water and glycerol channels (aquaglyceroporins or AQPs) to melarsoprol and pentamidine cross-resistance. Further analysis indicated a central role for one particular gene, AQP2, in cross-resistance, suggesting that natural resistance may be due to reduced AQP2 expression. There is a desperate need for research that improves our understanding of melarsoprol-pentamidine cross-resistance in African trypanosomes. Clearly, an improved understanding of the mechanisms involved will be important for human health, facilitating the development of diagnostic tools and improved therapies. In addition, an understanding of how aquaglyceroporins contribute to this phenomenon will make an important contribution to basic biomedical science. The proposal aims to improve our understanding of melarsoprol-pentamidine cross-resistance and to develop a diagnostic test. Specifically, the contribution of AQP2 to drug resistance will be assessed and exploited. The first key goal is to determine how different protein sequences in AQPs influence drug sensitivity. AQPs with different sequences will be assessed using genetic manipulation followed by drug resistance assays. This will allow drug sensitivity to be assigned to a specific protein sequence combination. These sequence combinations will then be similarly examined in AQPs found naturally to develop our understanding of the relationship between AQP function and drug resistance. The goal here is to predict and test whether natural trypanosome isolates are sensitive or resistant. Another set of experiments is designed to identify the protein sequences (zip-codes) that dictate the location of the AQPs in the cell and how this impacts on drug resistance. These results will allow us to predict the behavior of naturally occurring AQPs which will be tested experimentally. Trypanosomes that have no AQP genes will also be made. A variety of assays will then be used to assess the behavior of these cells and to address the question, are AQPs themselves promising drug targets? The new knowledge will finally be translated into a diagnostic test. The test will depend upon the use of an AQP2-specific antibody or a DNA-based diagnostic. A test for the presence of AQP2 will rapidly predict the melarsoprol-pentamidine sensitivity/resistance of clinical strains and allow for the selection of the most appropriate treatment.

African trypanosomes are transmitted by the tsetse fly and circulate in the bloodstream of their mammalian hosts. These protozoan parasites cause Human African Trypanosomiasis (HAT) and Nagana in livestock. HAT is typically fatal without chemotherapy but therapies have multiple problems, including toxicity and resistance. Commonly used HAT therapies are pentamidine, only suitable for pre-CNS disease, and melarsoprol for advanced disease. Melarsoprol treatment failure is a major problem and resistant trypanosomes often display pentamidine cross-resistance. Research lead by the applicant recently linked an unusual aquaglyceroporin, AQP2, to this phenotype. Thus, reduced AQP2 expression could explain innate or acquired resistance. Other unpublished data reveal that AQP2 is restricted to the flagellar pocket. The proposal now is to carry out a molecular dissection of the AQP2 sequences required for drug sensitivity. The AQP2 selectivity filter will be genetically dissected using an established set of strains and phenotype assays. Similar assays will be used to characterize a set of naturally occurring AQPs and their role in drug sensitivity. The findings will allow us to predict which AQPs confer drug sensitivity and which mutations lead to resistance. Since mechanisms of flagellar pocket sequestration are of more general interest and may be important for AQP2 function, the molecular determinants of sequestration will be similarly dissected. Identification of the molecular 'zip-codes' involved will allow us to predict subcellular localisation of naturally occurring AQPs. We will also generate a complete aqp null strain to gain insight into natural AQP function in trypanosomes. Our findings will then be translated into a diagnostic test that could rapidly reveal drug-resistant strains; an AQP2-based test that rapidly predicts the melarsoprol and pentamidine sensitivity/resistance phenotype of clinical strains will allow selection of the most appropriate treatment option.

The World Health Organization recently stated, "Given the achievements made in the control of sleeping sickness, elimination of the disease as a public health problem can be envisaged. Continuous and strenuous efforts are required to fight the disease and avoid its resurgence as experienced in the 1980s" (WHO Neglected tropical diseases, hidden successes, emerging opportunities). The primary objective of the current proposal is to advance our understanding of melarsoprol and pentamidine cross-resistance in African trypansosomes and to use this new knowledge to develop a diagnostic test for drug sensitive and resistant sleeping sickness. This could be a critical component of a strategy implemented to reduce the burden of disease in the coming years. Thus, the patients suffering from sleeping sickness will benefit from the research and the health systems and related organizations in endemic countries will also benefit.

The research will provide new knowledge and scientific advancement and addresses a key issue of importance to Africa. The findings may also be relevant to other trypanosomal disease with a more global distribution and will also deliver training to a highly skilled researcher. Although commercial exploitation is not a major driving force underlying most neglected tropical disease research, this particular proposal does have the potential to generate commercially exploitable results.