The power of network-based drug design and the interplay between metabolism and gene expression in Trypanosoma brucei

Westerhoff, H.V. [Promotor]Bakker, B.M. [Copromotor

Drug target identification through systems biology.

To rationalise drug target selection, we should understand the role of putative targets in biological pathways quantitatively. We review here how experimental and computational network-based approaches can aid more rational drug target selection and illustrate this with results obtained for microbes and for cancer. Comparison of the drug response of biochemical networks in target cells and (healthy) host cells can reveal network-selective targets

Dissecting the catalytic mechanism of Trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modelling.

Background: Trypanothione synthetase catalyzes the conjugation of spermidine with two GSH molecules to form trypanothione. Results: The kinetic parameters were measured under in vivo-like conditions. A mathematical model was developed describing the entire kinetic profile. Conclusion: Trypanothione synthetase is affected by substrate and product inhibition. Significance: The combined kinetic and modeling approaches provided a so far unprecedented insight in the mechanism of this parasite-specific enzyme. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites.

Peroxisomes of organisms belonging to the protist group Kinetoplastea, which include trypanosomatid parasites of the genera Trypanosoma and Leishmania, are unique in playing a crucial role in glycolysis and other parts of intermediary metabolism. They sequester the majority of the glycolytic enzymes and hence are called glycosomes. Their glycosomal enzyme content can vary strongly, particularly quantitatively, between different trypanosomatid species, and within each species during its life cycle. Turnover of glycosomes by autophagy of redundant ones and biogenesis of a new population of organelles play a pivotal role in the efficient adaptation of the glycosomal metabolic repertoire to the sudden, major nutritional changes encountered during the transitions in their life cycle. The overall mechanism of glycosome biogenesis is similar to that of peroxisomes in other organisms, but the homologous peroxins involved display low sequence conservation as well as variations in motifs mediating crucial protein-protein interactions in the process. The correct compartmentalisation of enzymes is essential for the regulation of the trypanosomatids' metabolism and consequently for their viability. For Trypanosoma brucei it was shown that glycosomes also play a crucial role in its life-cycle regulation: a crucial developmental control switch involves the translocation of a protein phosphatase from the cytosol into the organelles. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative of regulation of enzyme activities as an additional means to adapt the metabolic network to the different environmental conditions encountered. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann

Network-based selectivity of antiparasitic inhibitors.

Several diseases are caused by eukaryotic parasites, e.g. Malaria or African Sleeping Disease. One possible way to proceed against them is the knockout or an adequate inhibition of the parasite's metabolism by capable drugs, i.e. by inhibitors with a strong effect on the parasitic metabolism and a least possible effect in the host. The development of such drugs is complicated by the substantial problem of the biochemical similarity between the metabolisms of hosts and parasites. Especially in their essential parts, which are the most suited targets for the knockout, both metabolic systems are closely related. Therefore drug research in that field focuses on selectivity, i.e. capacity of an inhibiting drug to differentiate between host and parasite. Structure-based selectivity, which deals with differences in inhibitor binding as a result of the 3D-structure of the inhibited enzymes, dominates the interest of research, whereas network-based selectivity, which comprehends the properties of a metabolic network as they are described in Metabolic Control Theory, is almost neglected. The study of network-based selectivity should lead to a prediction of promising target enzymes for drugs as well as of the best suited inhibiting mechanism, e.g. competitive, non-competitive or uncompetitive inhibition.By the means of a newly defined term we have compared the selectivity of these different types of inhibitors for the first and the second step of a two-enzyme pathway. We also tried to generalize our findings for a linear pathway of arbitrary length. The method of comparing the flux control distribution or 'control profile' in a metabolic system of the parasite and the host for prediction of most effective targets for anti-parasitic drugs is called Differential Control Analysis (Bakker et al. 2000). The models of the glycolysis of the human erythrocyte (Schuster & Holzhütter 1995) and of Trypanosoma brucei (Bakker et al. 1997) are looked into in more detail

Control and regulation of gene expression: quantitative analysis of the expression of phosphoglycerate kinase in bloodstream form Trypanosoma brucei.

Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e.g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

Proliferating bloodstream- form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes.

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.3h, contained 46μmol of carbon (1