Using metabolomic analyses to study mode of action of and resistance to Eflornithine in Trypanosoma brucei

QR Microbiology;QD Chemistry

Vincent, Isabel May ; Burchmore, Richard

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: Trypanosoma, brucei, metabolomics, eflornithine, nifurtimox, DFMO, drug, mode of action, mechanism of resistance;   

Others  :  http://theses.gla.ac.uk/3125/1/2011vincentIphd.pdf

来源: University of Glasgow

【 摘 要 】

Human African trypanosomiasis (HAT) is a disease that is in desperate need of new pharmacological agents active against the causative parasite, the flagellated protozoan Trypanosoma brucei.In this thesis, new metabolomics techniques have been developed to study pathways in response to drug action with the aim of defining the mode of action of current and future drugs. Eflornithine, a polyamine pathway inhibitor, was used as a proof of principle, revealing both expected changes that correlate well with the literature and unexpected changes that lead to pathways and metabolites not previously described in bloodstream form trypanosomes. One metabolite not previously described in trypanosomes is acetylornithine, whose levels correlate well with ornithine and whose production comes directly from ornithine transported from the medium. Nifurtimox and the nifurtimox- eflornithine combination therapy were assayed for changes to their metabolomes revealing changes in nifurtimox treatment that included alterations to sugar and purine levels. The combination therapy had reduced changes to some metabolites compared to each drug in isolation suggesting reasons for the combination‟s lack of synergy. Isotopically labelled metabolites were also of use in determining flux through the pathways identified as being affected by drug perturbation. These techniques, along with other biochemical techniques, were used to show arginase activity is absent in bloodstream form trypanosomes and that ornithine is not made from arginine when ornithine is present in the medium. Arginine can, however, be used to produce ornithine through an arginase-independent mechanism when exogenous ornithine is lacking. Evidence is also provided that parts of the pentose phosphate pathway, not thought to be active in bloodstream form trypanosomes, may still be active in in vitro grown cells.A mechanism of resistance to eflornithine involving the deletion of an amino acid transporter that is able to transport eflornithine is also described. It is hoped that simple PCR-based tests for this resistance mechanism will be of use in resistant foci in prescribing appropriate drugs to HAT patients.