In Vivo and In Vitro Activity by Diverse Chelators against Trypanosoma brucei brucei1

A system of prescreens and screen has been developed to select chelators as potential drugs against Trypanosoma brucei brucei EATRO 110. The chelators tested were nearly all commercially available, low molecular, and having moderate to high affinity for Fe(III). We prescreened 70 compounds showing heme-sparing or inhibitory activity in a Crithidia fasciculata growth system having excess Fe and minimal hemin. Of these, 45 were highly trypanocidal for suspensions of bloodstream T. b. brucei; criteria of activity here were immobilization, lysis, and loss of infectivity. Eighteen of the chelators highly active in the suspension prescreen were tried in T. b. brucei-infected mice. Thirteen of these chelators were curative in mice with 24-h infections, that is, they allowed survival >30 days beyond the untreated controls. 3,4-Dihydroxycinnamic acid (caffeic acid). 2,9-dimethyl-1, 10 phenanthroline (neocuproine), and 2-pyridinecarboxaldehyde-2-pyridyl-hydrazone cured five out of five mice after an i.v. dose of 100 mg/kg. Salicylaldehyde thiosemicarbazone cured five out of five mice at an i.p. dose of 500 mg/kg. Lesser activity was shown by several other chelators.     

Identification of Three Classes of Heteroaromatic Compounds with Activity against Intracellular Trypanosoma cruzi by Chemical Library Screening

The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain) expressing β-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC₅₀: 54, 190 and 23 nM, respectively). Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti–T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC₅₀ values of 2 nM (PCH6 and CX2). These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.     

Identification of a telomeric DNA sequence in Trypanosoma brucei

A simple repetitive DNA sequence in the nuclear genome of Trypanosoma brucei, consisting of tandem repeats of the hexanucleotide 5' CCCTAA 3', was identified as being telomeric by several criteria. This sequence was specifically labeled with T. brucei genomic DNA as the template for in vitro nick translation by DNA polymerase I, and was present in Bal 31 nuclease sensitive, genomic restriction fragments of the large sizes expected in this organism for at least some telomeric regions. The same repeated sequence was found in six other flagellates tested. A segment of DNA from T. brucei including this telomeric sequence was cloned in pBR322 and characterized. The cloned segment contained a sequence highly homologous to the 3' ends of several variant surface glycoprotein mRNAs, upstream of the tandemly repeated hexanucleotide sequence.     

Ethyl Pyruvate Emerges as a Safe and Fast Acting Agent against Trypanosoma brucei by Targeting Pyruvate Kinase Activity

Background

Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties.

Results

The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0±0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions.

Conclusion

Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemolymphatic as well as neurological stages of sleeping sickness.     

Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei expression library by polymerase chain reaction

Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.     

Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

Identification of a developmentally regulated calcium-binding protein in Trypanosoma brucei

Calcium ions mediate cellular activity by binding to specific cellular proteins. The following study systematically examines the cellular complement of calcium-binding proteins in different cell fractions and life cycle stages of Trypanosoma brucei. Using a 45Ca-gel overlay procedure, eight calcium-binding proteins were consistently observed. The majority of proteins were cytosolic (84, 70, 64, 22, and 15 kd) while the remainder (55, 46, and 29 kd) were particulate. Although calmodulin was detected amongst the calcium-binding proteins, it did not represent the majority of calcium-binding activity. Of special interest was the 46 kd calcium-binding protein which was associated with 3-fold more calcium in cultured procyclic forms than in slender bloodstream forms. By contrast, promastigote forms of Leishmania mexicana did not contain the 46 kd calcium-binding protein. These data suggest that responsiveness to calcium signals may vary during the trypanosome life cycle as a result of changes in the cellular complement of calcium-binding proteins.     

Identification of a Developmentally Regulated Calcium-Binding Protein in Trypanosoma brucei1

Calcium ions mediate cellular activity by binding to specific cellular proteins. The following study systematically examines the cellular complement of calcium-binding proteins in different cell fractions and life cycle stages of Trypanosoma brucei. Using a ⁴⁵Ca-gel overlay procedure, eight calcium-binding proteins were consistently observed. The majority of proteins were cytosolic (84, 70, 64, 22, and 15 kd) while the remainder (55, 46, and 29 kd) were particulate. Although calmodulin was detected amongst the calcium-binding proteins, it did not represent the majority of calcium-binding activity. Of special interest was the 46 kd calcium-binding protein which was associated with 3-fold more calcium in cultured procyclic forms than in slender bloodstream forms. By contrast, promastigote forms of Leishmania mexicana did not contain the 46 kd calcium-binding protein. These data suggest that responsiveness to calcium signals may vary during the trypanosome life cycle as a result of changes in the cellular complement of calcium-binding proteins.     

Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-amino-acid C-terminal tail

Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2-/- procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 +/- 0.02 mM and 0.28 +/- 0.1 mM, respectively. The k(cat) value for TbHK1 is 2.9 x 10(4) min(-1). TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 microM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.     

Immunization with a tubulin-rich preparation from Trypanosoma brucei confers broad protection against African trypanosomosis

Tubulin from Trypanosoma brucei was purified to near homogeneity using a protocol which involved treatment with urea with subsequent renaturation and was then used to immunize mice. Renatured tubulin further purified by SDS-PAGE (denatured), synthetic tubulin peptides (STP), and rat brain tubulin (RbTub) were also used. Immunized mice were challenged with T. brucei, Trypanosoma congolense or Trypanosoma rhodesiense. Renatured T. brucei tubulin (nTbTub) induced protection in all mice tested, of which 60-80% (n = 81) was complete and the remainder partial. Denatured T. brucei tubulin (dTbTub), STP, or RbTub induced lower antibody levels than nTbTub and did not offer protection. However, in culture, the antibodies against dTbTub or STP killed trypanosomes although at lower dilutions than nTbTub, but those against RbTub did not. In Western blots anti-trypanosome antibodies recognized the tubulin of all the trypanosome species investigated but not vertebrate tubulin, whereas the anti-RbTUB antibodies recognized both trypanosome and vertebrate tubulin. Of the five mice given passive immunity by the transfer of anti-nTbTub serum, four were completely protected and one partially protected. These data suggest that tubulin is the relevant immunogen in the preparation used and could therefore be a promising target for the development of a parasite-specific, broad spectrum vaccine.     

Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein

The variant surface glycoprotein (VSG) of Trypanosoma brucei has a glycolipid covalently attached to its C terminus. This glycolipid, which anchors the protein to the cell membrane, is attached to the VSG polypeptide within 1 min after translation (Bangs, J. D. Hereld, D., Krakow, J.L., Hart, G. W., and Englund, P. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3207-3211). This rapid processing suggests that, prior to incorporation, the glycolipid may exist in the cell as a preformed precursor which is transferred to the VSG polypeptide en bloc. We have isolated a molecule which has properties consistent with it being a VSG glycolipid precursor. It is highly polar and can be labeled by [3H] myristate but not by [3H]palmitate. It reaches steady state during continuous labeling with [3H]myristate and shows rapid turnover in pulse-chase experiments, suggesting that it is a metabolic intermediate rather than an end product. When treated with HNO2 it liberates phosphatidylinositol, as does VSG (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Also, like VSG, it releases a compound which co-migrates on thin layer chromatography with dimyristylglycerol when treated with the purified endogenous phospholipase C from trypanosomes. After treatment with this lipase, the putative precursor can be immunoprecipitated by antibodies directed against the C-terminal cross-reactive antigenic determinant of the VSG. These data provide strong evidence that this glycolipid is a VSG precursor.     

Acaricidal Activity of Eugenol Based Compounds against Scabies Mites

Backgound

Human scabies is a debilitating skin disease caused by the “itch mite” Sarcoptes scabiei. Ordinary scabies is commonly treated with topical creams such as permethrin, while crusted scabies is treated with topical creams in combination with oral ivermectin. Recent reports of acaricide tolerance in scabies endemic communities in Northern Australia have prompted efforts to better understand resistance mechanisms and to identify potential new acaricides. In this study, we screened three essential oils and four pure compounds based on eugenol for acaricidal properties.

Methodology/Principal Findings

Contact bioassays were performed using live permethrin-sensitive S. scabiei var suis mites harvested from pigs and permethrin-resistant S. scabiei var canis mites harvested from rabbits. Results of bioassays showed that clove oil was highly toxic against scabies mites. Nutmeg oil had moderate toxicity and ylang ylang oil was the least toxic. Eugenol, a major component of clove oil and its analogues –acetyleugenol and isoeugenol, demonstrated levels of toxicity comparable to benzyl benzoate, the positive control acaricide, killing mites within an hour of contact.

Conclusions

The acaricidal properties demonstrated by eugenol and its analogues show promise as leads for future development of alternative topical acaricides to treat scabies.     

Kola acuminata proanthocyanidins: a class of anti-trypanosomal compounds effective against Trypanosoma brucei

Human African trypanosomiasis is undergoing an alarming rate of recrudescence in many parts of sub-Saharan Africa. Yet, there is no successful chemotherapy for the disease due to a limited number of useful drugs, side effects and drawbacks of the existing medication, as well as the development of drug resistance by the parasite. Here we describe a new lead anti-trypanosomal compound isolated from Kola acuminata (Makasu). We purified a proanthocyanidin by chromatographic procedures and confirmed its homogeneity and structure by Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionisation Time-of-Flight mass spectrometry, respectively. In vitro, this compound potently induced growth arrest and lysis of bloodstream form trypanosomes in a dose- and time-dependent manner. In a mouse model, it exhibited a trypanostatic effect that extended the life of infected, treated animals up to 8 days post-infection against the 4 days for infected, untreated animals. The proanthocyanidin showed a low cytotoxicity against mammalian cells, whereas treated-BF showed massive enlargement of their flagellar pocket and lysosome-like structures caused by an intense formation of multivesicular bodies and vesicles within these organelles. The observed ultrastructural alterations caused rupture of plasma membranes and the release of cell contents, indicative of a necrotic process rather than a programmed cell death. Interestingly, the proanthocyanidin acted against BF but not procyclic form trypanosomes. This new anti-trypanosomal compound should be further studied to determine its efficacy and suitability as an anti-trypanosomal drug and may be used as a tool to define novel specific drug targets in BF trypanosomes.     

Stage-specific requirement of a mitogen-activated protein kinase by Trypanosoma brucei

In cycling between the mammalian host and the tsetse fly vector, African trypanosomes undergo adaptive differentiation steps that are coupled to growth control. The signaling pathways underlying these cellular processes are largely unknown. Mitogen-activated protein kinases (MAPKs) are known mediators of growth and differentiation in other eukaryotic organisms. To establish the function of a MAPK homologue, TbMAPK2, in T. brucei, a null mutant was constructed. Bloodstream forms of a deltamapk2/deltamapk2 clone were able to grow normally and exhibited no detectable phenotype. When these cells were triggered to differentiate in vitro, however, they developed to the procyclic (fly midgut) form with delayed kinetics and subsequently underwent cell cycle arrest. Introduction of an ectopic copy of the TbMAPK2 gene into the null mutant restored its ability to differentiate and to divide. In contrast, a TbMAPK2 mutant, in which the T190 and Y192 residues of the activating phosphorylation site were replaced by A and F, was unable to restore the growth and differentiation phenotypes. Analysis of the DNA content and the nucleus/kinetoplast configuration of individual cells showed that the null mutant was arrested in all phases of the cell cycle and that 25-30% of the cells had failed to segregate their nucleus and kinetoplast correctly. This implies that cell cycle progression by the procyclic form depends on a constitutive stimulus exerted by the signaling cascade operating through TbMAPK2.     

Untargeted metabolomics reveals a lack of synergy between nifurtimox and eflornithine against Trypanosoma brucei

A non-targeted metabolomics-based approach is presented that enables the study of pathways in response to drug action with the aim of defining the mode of action of trypanocides. Eflornithine, a polyamine pathway inhibitor, and nifurtimox, whose mode of action involves its metabolic activation, are currently used in combination as first line treatment against stage 2, CNS-involved, human African trypanosomiasis (HAT). Drug action was assessed using an LC-MS based non-targeted metabolomics approach. Eflornithine revealed the expected changes to the polyamine pathway as well as several unexpected changes that point to pathways and metabolites not previously described in bloodstream form trypanosomes, including a lack of arginase activity and N-acetylated ornithine and putrescine. Nifurtimox was shown to be converted to a trinitrile metabolite indicative of metabolic activation, as well as inducing changes in levels of metabolites involved in carbohydrate and nucleotide metabolism. However, eflornithine and nifurtimox failed to synergise anti-trypanosomal activity in vitro, and the metabolomic changes associated with the combination are the sum of those found in each monotherapy with no indication of additional effects. The study reveals how untargeted metabolomics can yield rapid information on drug targets that could be adapted to any pharmacological situation.     

Inhibitors of human histone deacetylase with potent activity against the African trypanosome Trypanosoma brucei

A number of hydroxamic acid derivatives which inhibit human histone deacetylases were investigated for efficacy against cultured bloodstream form Trypanosoma brucei. Three out of the four classes tested displayed significant activity. The majority of compounds blocked parasite growth in the submicromolar range. The most potent was a member of the sulphonepiperazine series with an IC(50) of 34nM. These results identify lead compounds with potential for the development of a novel class of trypanocidal agent.