Sleeping sickness, caused by Trypanosoma brucei, is a deadly disease that affects some of the poorest countries in sub-Saharan Africa. Although the disease prevalence is declining, strengthening of the current control efforts, including introduction of more adequate chemotherapeutic options, is needed to prevent the re-emergence of yet another epidemic. Nitroaromatic compounds, such as nifurtimox (in combination with eflornithine) and fexinidazole (in clinical trials), have been recently introduced for the treatment of the second stage of sleeping sickness. These compounds are believed to act as pro-drugs that require intracellular enzymatic activation for antimicrobial activity. Here, the role of the bacterial-like nitroreductase TbNTR as a nitrodrug activating enzyme is examined through overexpression and knock-out studies in T. brucei. Multiple attempts to purify soluble recombinant TbNTR from E. coli were unsuccessful, because the recombinant protein was found to be membrane associated. In keeping with the role of TbNTR in nitrodrug activation, loss of an NTR gene copy in T. brucei was found to be one, but not the only, mechanism that may lead to nitrodrug resistance. Furthermore, in the bloodstream form of T. brucei, resistance was relatively easy to select for nifurtimox, with no concurrent loss of virulence and at clinically relevant levels. More worryingly, nifurtimox resistance led to a decreased sensitivity of these parasites to other nitroaromatic compounds, including a high level of cross-resistance to fexinidazole. Conversely, generation of fexinidazole resistance resulted in cross-resistance to nifurtimox. Should these findings translate to the field, emerging nitrodrug resistance could reverse all recent advances in the treatment of sleeping sickness, made since the introduction of eflornithine 20 years ago. Therefore, all efforts should be made to ensure nitroaromatic drugs are used only in drug combination therapies against sleeping sickness, in order to protect them from emerging resistance.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains. T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other. / Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Myo-inositol is ubiquitous in nature and is found at the structural core of a diverse range of biologically important derivatives, including phosphatidylinositols, inositol phosphates and mycothiol. The synthesis of myo-inositol derivatives is notoriously difficult due to the need to control both regio- and enantioselectivity. As a result, synthetic routes to derivatives of this type are often lengthy and low yielding. The first biosynthetic step in the production of all myo-inositol metabolites is the isomerisation of D-glucose 6- phosphate to L-myo-inositol 1-phosphate as mediated by L-myo-inositol 1-phosphate synthase (INO1). For the protozoan parasite Trypanosoma brucei, INO1 is essential for survival and its version of the enzyme (TbINO1) has a high turnover. This makes TbINO1 an attractive candidate for the biocatalytic production of L-myo-inositol 1- phosphate, and a potential starting point for drastically shortened syntheses of important myo-inositol derivatives. The production of L-myo-inositol 1-phosphate by TbINO1 has been optimised to achieve complete conversion in reaction conditions that facilitate product isolation. Due to problems with an in-batch process, the TbINO1 enzyme was immobilised and the process was transferred to a flow system. This has allowed for production of significant quantities of L-myo-inositol 1-phosphate with a high level of purity. L-myo-inositol 1- phosphate obtained from the flow system has been used to prepare mycothiol glycosylation acceptor, 1,2,4,5,6-penta-O-acetyl-D-myo-inositol, in a concise synthesis with a greatly improved yield over the literature.

One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.

No description available.

No description available.

No description available.

Trypanosoma brucei, the causative agent of African trypanosomiasis, and its relatives Trypanosoma cruzi and several Leishmania species belong to a class of protozoa called kinetoplastids that cause a significant health burden in tropical and semitropical countries across the world. While an improved therapy was recently approved for African trypanosomiasis, the therapies available to treat infections caused by T. cruzi and Leishmania spp. remain relatively poor. Improving our understanding of T. brucei metabolism can inform on metabolism of its relatives. The purpose of the research presented in this dissertation was to develop novel tools and methods to study metabolism in T. brucei with the ultimate aim to improve treatments of all kinetoplastid diseases. We developed a novel tool to study glycosomal pH in the bloodstream form of T. brucei. Using this tool, we discovered that this life stage regulates glycosomal pH differently than the procyclic form, or insect-dwelling stage, and only uses sodium/proton transporters to regulate glycosomal pH. I pioneered a thermal proteome profiling method in this parasite to discover drug targets and their effects on cell pathways. Using this method, I found that other proteins may be involved in glycosomal pH regulation, including PEX11 and a vacuolar ATPase. This method also illuminated several important pathways influenced by glycosomal pH regulation, including glycosome proliferation, vesicle trafficking, protein glycosylation, and amino acid transport. Metabolic studies in kinetoplastid parasites are currently hampered by the lack of available chemical probes. We developed a novel flow cytometry-based high-throughput drug screening assay to discover chemical probes of T. brucei glycolysis. This method combines the advantages of phenotypic (or cell-based) screens with the advantage of targeted (purified protein) screens by multiplexing biosensors that measure multiple glycolytic metabolites simultaneously, such as glucose, ATP, and glycosomal pH. The complementary information gained is then used to distinguish the part of glycolysis identified inhibitors target. We validated the method using the well characterized glycolytic and alternative oxidase inhibitors 2-deoxyglucose and salicylhydroxamic acid respectively. We demonstrated the screening assay with a pilot screen of 14,976 compounds with decent hit rates for each sensor (0.2-0.4%). About 64% of rescreened hits repeated activity in at least one sensor. We demonstrated one compound with micromolar activity against two biosensors. In summary, we developed and demonstrated a novel screening method that can discover glycolytic chemical probes to better study metabolism in this and related parasites. There are few methods to study enzyme kinetics in the live-cell environment. I developed a kinetic flow cytometry assay that can measure enzyme and transporter activity using fluorescent biosensors. I demonstrated this by measuring glucose transport kinetics and alternative oxidase inhibition kinetics, with the measured kinetic parameters similar to those previously reported. We plan to expand on this method to measure transport kinetics in the glycosome and other organelles which has not been done before. We previously performed a drug screen to identify inhibitors that decrease intracellular glucose in T. brucei. I have performed preliminary work identifying the glucose transporter THT1 as one of the targets of optimized glucose inhibitors using the previously mentioned thermal proteome profiling method. We expect this finding will improve our ability to move these compounds from hit to lead in the drug discovery pipeline. Together, I have developed several flow cytometry and proteomics methods to better study metabolism in T. brucei. These tools are beginning to be used in related parasites. We expect the discoveries made using these tools will improve our ability to treat these neglected tropical diseases.

Human African trypanosomiasis (sleeping sickness) is a devastating disease which is endemic in parts of sub-Saharan Africa. It is caused by the protozoan parasite T. brucei, which are transmitted by the bite of infected tsetse flies. Although the disease is fatal if left untreated, there is a lack of safe, effective and affordable drugs available; therefore new drugs are urgently needed. The aim of the work presented in this thesis is to develop novel trypanocidal compounds. It is divided into two parts to reflect the two distinct strategies employed to achieve this aim. The first part focuses on the inhibition of glycophosphoinositol (GPI) anchor synthesis by inhibiting the Zn2+-dependent enzyme, GlcNAc-PI de-N-acetylase. Trypanosomes have a variable surface glycoprotein (VSG) coat, which allows them to evade the human immune system. The GPI anchor attaches the VSG to the cell membrane; therefore inhibiting GPI synthesis should expose the parasite to the immune system. Initially, large substrate analogues were synthesized. These showed weak inhibition of the enzyme. Zinc-binding fragments were screened, and small molecule inhibitors based on salicylhydroxamic acid were then synthesized. These compounds showed modest inhibition, but the excellent ligand efficiency of salicylhydroxamic acid indicates this may be a promising starting point for further inhibitors. The second part details the P2 strategy. The P2 transporter is a nucleoside transporter unique to T. brucei, which concentrates adenosine. The transporter also binds and selectively concentrates compounds that contain benzamidine and diaminotriazine P2 motifs, which can enhance the potency and selectivity of these compounds. The sleeping sickness drugs melarsoprol and pentamidine contain P2 motifs. Compounds comprising a P2 targeting motif, a linker and a trypanocidal moiety were synthesized. Initially, a diaminotriazine P2 motif was attached to a trypanocidal tetrahydroquinoline (THQ) protein farnesyl transferase (PFT) inhibitor, with limited success. The P2 strategy was also applied to a non-selective, trypanocidal, quinol moiety. The quinol moiety was attached to diaminotriazine and benzamidine P2 motifs, and an increase in selectivity for T. brucei over MRC5 cells was observed.