Drug resistance in African trypanosomes

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

African sleeping sickness reached epidemic proportions in the 1990s, with an estimated 500,000 people infected and millions at risk in 36 African countries. The disease is caused by a parasite called a trypanosome, transmitted by bites of the tsetse fly, and is fatal if not treated. Available drugs are very old, introduced in the 1940s. The early stage, with parasites living freely in the blood, can be effectively treated with pentamidine but treatment can have serious side effects and has to be given through multiple injections in hospital. The treatment for the late stage, in which the parasite has entered the brain and causes severe neurological symptoms ending in coma and death, is melarsoprol - a drug based on arsenic and so toxic that it causes brain damage in up to 10% of patients, killing up to half of these. The only alternative treatment, eflornithine, is very expensive and requires four daily intravenous infusions for up to two weeks ? often impossible in rural Africa. A further problem is that the parasite has developed resistance to melarsoprol, leading to high levels of treatment failure, risking arsenic-induced brain damage for nothing. Newer drugs are in development but these are of the same class as pentamidine, known as diamidine drugs, and parasites resistant to melarsoprol are often cross-resistant to diamidines as well.
We have studied melarsoprol and diamidine resistance in these parasites and found that it is caused by the loss of particular proteins, embedded in the parasites surface membrane, that facilitate the uptake of very specific nutrients by the organism. One such protein, P2, is responsible for uptake of the essential compound adenosine but both diamidines and melarsoprol use this conduit to enter the trypanosome as well. Mutations in this transporter lead to mild resistance to both classes of drugs but we recently found that high levels of resistance would require the further loss of a second transport protein, HAPT, while some diamidines can further enter the parasite through yet a third protein, LAPT. The aim of the proposed research project is to identify the genes for these new transporters so that it can be assessed whether mutations in these genes can indeed be linked to resistance. We further want to study the transporters in sufficient detail to allow chemists to make custom-designed drugs that will use all three transporters, making the new generation of medicines more effective and circumventing resistance.

Technical Summary

Human African trypanosomiasis, better known as sleeping sickness, continues to be a scourge of rural Africa. By far the highest incidence is caused by tsetse fly-transmitted infection with the protozoan parasite Trypanosoma brucei gambiense, which causes West-African sleeping sickness. This is a chronic disease that is believed to have a near 100% fatality rate unless adequately treated. In the early stage of the disease the parasite is present in the haemo-lymphatic system and this stage is treated with the diamidine drug pentamidine. In the late stage the parasite has penetrated the blood-brain barrier and infiltrated the central nervous system, causing the characteristic neurological symptoms. This stage is usually treated with the arsenic-containing drug melarsoprol, except in some areas with particularly high incidence of treatment failure where difluormethyl ornithine (DFMO) has been introduced as a replacement.
The very high rate of melarsoprol treatment failure, occurring in geographically distant foci, as well as the well-documented phenomenon of arsenical-diamidine cross-resistance is a genuine threat to the control of this disease, particularly since the only new treatment in development, furamidine, is also a diamidine. We have studied diamidine and arsenical resistance in African trypanosomes and found that this is caused by changes in plasma membrane transporters, leading to reduced uptake of the drug by the parasite. Low level resistance to both pentamidine and melarsoprol are observed when the TbAT1 gene, encoding the P2 aminopurine transporter, is deleted. Much higher levels of resistance to both drugs are in evidence upon subsequent loss of a second transporter, the High Affinity Pentamidine Transporter (HAPT). A third diamidine transporter, the Low Affinity Pentamidine Transporter, does not appear to be involved in arsenical uptake.
While the identity of the P2 transporter gene is known and specific mutations have been tentatively linked to melarsoprol resistance, the genes encoding HAPT and LAPT are not known. The primary aim of this proposal is to identify and clone these genes and express them in a suitable system for further characterisation. Identification of the genes will enable us to assess (1) their status in drug resistant laboratory strains and field isolates and (2) their physiological role in the parasite. We further aim to study the substrate selectivity of these transporters in detail, constructing a model for substrate recognition that will inform the synthesis of improved diamidine drugs that are efficiently taken up trough multiple transporters including LAPT and thus avoid cross-resistance with melarsoprol.

Publications

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Baker N (2012) Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes. in Proceedings of the National Academy of Sciences of the United States of America

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Barrett MP (2010) Potential new drugs for human African trypanosomiasis: some progress at last. in Current opinion in infectious diseases

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Delespaux, V And De Koning HP (2013) Trypanosomatid disease: molecular routes to drug discovery

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Garcia-Salcedo J (2014) Trypanosomes and Trypanosomiasis

 
Description New diamidine drugs for animal trypanosomiasis
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in advisory committee
Impact Consultation meeting by GALVmed with experts on drugs for African trypanosomiasis, and drug resistanc e mechanism, to set priorities and strategy towards new drug development for this disease. I presented research and opinions on this subject and as a redult of my work on drug resitance, the then-ptreferred candidate for further development was dropped, as we showed cross-resistance with the therapeuitic agents it has being developed to replace. An alternative strategy was adopted.
 
Description Invited to apply through personal contact
Amount £155,577 (GBP)
Organisation GALVmed 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 09/2013
 
Description Travel/training grant
Amount £1,400 (GBP)
Organisation Boehringer Ingelheim 
Sector Private
Country Germany
Start 01/2011 
End 05/2011
 
Description Wellcome Trust studentship
Amount £111,303 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 10/2016
 
Description joint International Project
Amount £11,500 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2009 
End 02/2011
 
Title Leishmania and Crithidia cell lines expressing Trypanosoma drug transporters 
Description Standardised clonal cell lines of the kinetoplastid species Leishmania major and Crithidia fasciculata have been transformed with genes encoding potential drug transporters from Trypanosoma brucei brucei. 
Type Of Material Cell line 
Provided To Others? No  
Impact These cell lines have very recently been generatged and it is hoped they will provide the final evidence of the identity of the transporter genes; analysis of the transformatnts is in progress. 
 
Title Yeast cell lines expressing T. brucei resistance markers 
Description Yeast cell lines have been generated to express wild-type or various mutated forms of the resistance genes under study. 
Type Of Material Cell line 
Provided To Others? No  
Impact The study of each transporter gene in isolation in the yeast system is providing extremely useful insights into the structure and the function of these transporters. 
 
Title Yeast growth assay to screen for functional mutations in T. brucei drug transporters 
Description Yeast cell lines have been generated that express various WT and mutated T. brucei drug transporters. An assay has been optimised for screening theses cell ines ,and thus various transporter mutations, for increasing the sensitivity of the yeast lines towards various trypanocides. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This assay allows us to rapidly screen for functional mutations in individual T. brucei drug transporters. These mutations may contribute to observed resistance phenotypes 
 
Title database of inhibition profiles for diamidine transporters in trypanosoma brucei 
Description Dataset of inhibition constant and other data for the purpose of establishing a complete structure/activity relationship for each of the diamidine transporters. 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2009 
Provided To Others? Yes  
Impact A manuscript on the analysis of the data will be prepared. The anaylsis will provide insights in how drug transporters work and what the molecular basis for their selectivity is. A predictive model for drug uiptake in this pathogen should ensue. 
 
Title production of new genetically modified parasites as a model for studying drug resistance in sleeping sickness 
Description Production of new vectors, genetically modified clonal lines and drug-adapted resistant lines 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Essential material for understanding diamidine and arsenical resistance in human sleeping sickness. This is leading to new insights into the causes of resistance as well as into the possibility of reversal agents and new drugs. 
 
Description Expresssion of a novel resistance determinant in yeast 
Organisation Central University of Venezuela
Department Escuela de bioanalisis
Country Venezuela, Bolivarian Republic of 
Sector Academic/University 
PI Contribution We have expressed both wild-type and novel mutant copies of the gene under study in the specialised yeast strains using the vector provided by the collaborator.
Collaborator Contribution Plasmids containing WT copies of the genes of interest, in a suitable expression vector, were provided free of charge. Technical advise and experimental protocols were also provided.
Impact strains have been generated and techniques optimised; experimental phase has almost completed and a manuscript outline has been discussed in detail with the collaborators.
Start Year 2010
 
Description Identification and validation of a new resistance determinant for in Trypanosoma brucei 
Organisation London School of Hygiene and Tropical Medicine (LSHTM)
Department Faculty of Infectious and Tropical Diseases
Country United Kingdom 
Sector Academic/University 
PI Contribution We have extablished that the potential drug resistance mechanism identified from a primary screening process is indeed able to induce resistance and found an inactive copy of the gene in a highly resistant strain. We have now cloned both WT and altered genes into a suitable expression system for further analysis. A Master student is working on this.
Collaborator Contribution From a genome-wide screen a new drug resistance determinant was identified. Because we are funded by the MRC to investigate drug resistance mechanisms in trypanosomes, this information was confidentially made available to us and we were able to validate this primary observation. We have following this upand this has led to multiple research papers, conference presentations etc.. Research papers: Baker N, Glover L, Munday JC, Aguinaga Andrés D, Barrett MP, De Koning HP, and Horn D (2012) Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes. Proc Natl Acad Sci USA 109:10996-11001 Munday JC, Eze AA, Baker N, Glover L, Clucas C, Aguinaga Andrés D, Natto MJ, Teka IA, McDonald J, Lee RS, Graf FE, Ludin P, Burchmore RJS, Turner CMR, Tait A, MacLeod A, Mäser P, Barrett MP, Horn D, and De Koning HP (2014) Trypanosoma brucei Aquaglyceroporin 2 is a high affinity transporter for pentamidine and melaminophenyl arsenic drugs and is the main genetic determinant of resistance to these drugs. J Antimicrob Chemother 69:651-663. Graf FE, Baker N, Munday JC, De Koning HP, Horn D, and Mäser P (2015) Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates. Int J Parasitol Drugs Drug Res 5:65-68 Graf FE, Ludin P, Arquint C, Schmidt RS, Schaub N, Kunz-Renggli C, Munday JC, Krezdorn J, Baker N, Horn D, Balmer O, Caccone A, De Koning HP and Mäser P (2016) Comparative genomics of drug resistance of the sleeping sickness parasite Trypanosoma brucei rhodesiense. Cell Mol Life Sci 73:3387-3400 Reviews and Opinion pieces: Baker N, De Koning HP, Mäser P, and Horn D (2013) Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol 29:110-118 115. Zoltner M, Horn D, De Koning HP, Field MC (2016) Exploiting the Achilles' heel of membrane trafficking in trypanosomes. Curr Opin Microbiol 34:97-103
Impact The collaborator has used the preliminary data generated from a Wellcome Trust grant on which we are named collaborators. This was recently awarded. Update 2011: Much progress has been made to understand the contribution of this new drug resistance marker. This project is nearing completion. Update 2012: 1 major paper published with both PI's (Horn and De Koning) listed as joint corresponding authors (Proc. Natl Acad Sci USA 2012); a further and much larger paper is nearing completion as of Nov 2012. De Koning will be senior and corresponding author on this one. Both papers are the dirct result of the MRC award to which this is linked. Update: the larger paper was published in 2014 (J Antimicrob Chemother 69:651-663). Further this collaboration.
Start Year 2010
 
Description Identification of mutant Aquaporin genes in clinical isolates of African Trypanosomiasis 
Organisation Swiss Tropical & Public Health Institute
Department Parasite Chemotherapy
Country Switzerland 
Sector Public 
PI Contribution The MRC grant G0701258 on identifying novel transporter genes involved in Pentamidine and arsenical resistance identified Trypanosoma brucei Aquaporin 2 as a key determinant of drug resistance.
Collaborator Contribution The Swiss group confirmed deletions in this gene in some of their resistant strains and in clinical field isolates of relapse patients.
Impact One PhD thesis , several research papers and conference presentations were the result of this collaboration as listed below: Graf FE, Ludin P, Wenzler T, Kaiser M, Pyana P, Büscher P, De Koning HP, Horn D, and Mäser P (2013) Aquaporin 2 mutations in Trypanosoma b. gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol. PLoS Negl Trop Dis 7:e2475 Graf FE, Baker N, Munday JC, De Koning HP, Horn D, and Mäser P (2015) Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates. Int J Parasitol Drugs Drug Res 5:65-68 Graf FE, Ludin P, Arquint C, Schmidt RS, Schaub N, Kunz-Renggli C, Munday JC, Krezdorn J, Baker N, Horn D, Balmer O, Caccone A, De Koning HP and Mäser P (2016) Comparative genomics of drug resistance of the sleeping sickness parasite Trypanosoma brucei rhodesiense. Cell Mol Life Sci 73:3387-3400
Start Year 2011
 
Description Inhibitors for diamidine transporters 
Organisation Spanish National Research Council (CSIC)
Country European Union (EU) 
Sector Public 
PI Contribution Testing for inhibitory activity on diamidine transporters of Trypanosoma brucei
Collaborator Contribution supply of unique chemical compounds of great value to the MRC grant objectives, that were not commercially avaliable
Impact To date we have obtained International Joint Project funding from the Royal Society for this collaboration (value £11,500) for mutual visits and limited research expenditure. Compounds have ben supplied by the collaborator and have been tested by ourselves, generating a growing dataset. Initial inhibitors have been identified. It emerges that some of the inhibitors are powerful trypanocides in their own right. This is an unexpected outcome and will in due course be the subject of a new project application. A self-funded PhD student is investigating the mechanism by which these compounds kill trypanosomes. Update 2011: this work on the mechanism of action of these drug transporter inhibitors is now almost complete and is being prepared for publication.
Start Year 2008
 
Description Mechanism of action of phosphonium compounds in Trypanosoma species 
Organisation Academy of Sciences of the Czech Republic
Country Czech Republic 
Sector Learned Society 
PI Contribution We have established the activity of a series of phosphonium compounds against kinetoplastid parasites and conducted studies on their mechanism of action, showing that they target trypanosome mitochondria
Collaborator Contribution Zikova's group have been performing detailed biochemical assays to pinpoint the exact mechanism of action with in the mitochondria.
Impact This has already contributed to one PhD thesis, successfully defended in October 2012. Research paper: Alkhaldi AAM, Martinek J, Panicucci B, Dardonville C, Ziková A, and de Koning HP (2016) Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei. Int J Parasitol Drugs Drug Res 6:23-34
Start Year 2012
 
Description QSAR of High Affinity Pentamidine Transporter 
Organisation Shanghai Jiao Tong University
Country China 
Sector Academic/University 
PI Contribution As part of the MRC-funded programme we have evaluated a large number of substrates for inhibition of the pentamidine transporters. This has resulted in a data set of approximately 74 structures with associated inhibition constants for the High Affinity Pentamidine Transporter (HAPT) alone. This dataset is now being used for Quantitative Structure-Activity Relationship (QSAR) analysis using a CoMSIA approach by new collaborators at the Department of Chemistry of the university of Cambridge, specifically Dr Fredrik Svensson.
Collaborator Contribution 3D computerised modelling of ligand interactions with the binding pocket of the drug transporter under investigation.
Impact A preliminary model based on part of the dataset has been generated. The full model is under construction and is anticipated to lead to a fine publication in a medium impact Journal. Update 2017: Model completed; drafting of paper started.
Start Year 2016
 
Description structure-activity relationships of diamidine transporters 
Organisation University of North Carolina at Chapel Hill
Country United States 
Sector Academic/University 
PI Contribution Assessing a number of diamidine compounds for inhibition of certain transport proteins in trypanosoma brucei
Collaborator Contribution Supply free of charge of chemical compounds essential to the research objectives of the MRC grant. These chemicals are unique and are not commercially available.
Impact To date, data have been generated, contributing to a fuller understanding of the problem being investigated but no publication or conference proceedings have yet been produced as the work is ongoing. 2011 update: experimental phase of generating data has been successfully concluded and the data is now being used to construct a comprehensive computer-generated 3D QSAR model in collaboration with Shanghai Jiaotong University. 2012 update paper being drafted
Start Year 2009