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Trypanosoma brucei rhodesiense was named after Rhodesia which, in turn, was named after the British imperialist and white supremacist Cecil Rhodes. In the light of the Black Lives Matter movement and contemporary consciousness of postcolonial legacy, it seems opportune to reconsider the subspecies name. Pros and cons of renaming T. b. rhodesiense are discussed. [ABSTRACT FROM AUTHOR]

As a group of biologically active compounds, polyether antibiotics (ionophores) show a broad spectrum of interesting pharmacological properties, ranging from anti-bacterial to anti-cancer activities. There is increasing evidence that ionophores, including salinomycin (SAL), and their semi-synthetic analogues are promising candidates for the development of drugs against parasitic diseases. Our previous studies have shown that esterification and amidation of the C1 carboxylate moiety of SAL provides compounds with potent activity against Trypanosoma brucei , protozoan parasites responsible for African trypanosomiasis. In this paper, we present the synthetic pathways, crystal structures and anti-trypanosomal activity of C1 esters, amides and hydroxamic acid conjugates of SAL , its C20-oxo and propargylamine analogues as well novel C1/C20 doubly modified derivatives. Evaluation of the trypanocidal and cytotoxic activity using bloodstream forms of T. brucei and human myeloid HL-60 cells revealed that the single-modified C20-oxo and propargylamine precursor molecules 10 and 16 were the most anti-trypanosomal and selective compounds with 50% growth inhibition (GI 50) values of 0.037 and 0.035 μM, and selectivity indices of 252 and 300, respectively. Also the salicylhydroxamic acid conjugate of SAL (compound 9) as well as benzhydroxamic acid and salicylhydroxamic acid conjugates of 10 (compounds 11 and 12) showed promising trypanocidal activities with GI 50 values between 0.032 and 0.035 μM but less favorable selectivities. The findings confirm that modification of SAL can result in derivatives with improved trypanocidal activity that might be interesting lead compounds for further anti-trypanosomal drug development. Image 1 • A library of novel double-modified salinomycin (SAL) analogues was devised. • Compounds 14 and 15 were characterized using the scXRD method. • Two novel analogues, compounds 11 and 12 , showed promising trypanocidal activity. • Most derivatives showed lower cytotoxic activity against HL-60 cells than SAL. • Anti-trypanosomal mechanism of action of newly synthesized compounds was studied. [ABSTRACT FROM AUTHOR]

Abstract Despite some progress in recent years, the fight against parasitic diseases still remains a great challenge. Parasitic diseases affect primarily (but not exclusively) the poorest people living in underdeveloped regions of the world. The distribution of parasitoses are linked to tropical and subtropical climate conditions, to population growth and to impoverishment. If not treated, parasitic diseases may lead to serious health problems, and even death. Particularly vulnerable groups include infants and young children, pregnant women and immunocompromised individuals. Polyether ionophore antibiotics (ionophores), traditionally used in veterinary medicine as anti-coccidial feed additives and non-hormonal growth promoters, are of considerable interest, as they have been found to be highly effective agents against various parasites, both in vitro and in vivo. This review summarizes the anti-parasitic effects of the most important polyether ionophores against parasites that are responsible for a number of animal and human parasitic diseases. Recent findings and advances that support the potential of polyether ionophore antibiotics as novel anti-parasitic drug candidates are also presented and discussed. Graphical abstract Image 1 Highlights • Polyether ionophores have been found to be highly effective agents against various parasites, both in vitro and in vivo. • This review summarizes the anti-parasitic effects of the most important polyether ionophores against parasites. • Derivatives of ionophores show even better anti-parasitic activities compared to their unmodified parent compounds. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • some β2-specific inhibitors display moderate trypanocidal activity. • β2-specific inhibitors do not block proteasomal trypsin-like activity of T. brucei. • study proves inhibitor sensitivity differences between human/ T. brucei proteasome. Abstract Previous studies indicated that the proteasome of the protozoan parasite Trypanosoma brucei is particularly sensitive to inhibition of the trypsin-like activity. In this study, three newly developed β2 subunit-specific inhibitor (LU-102, LU-002c and LU-002i) were tested for their ability to block the trypsin-like activity of the trypanosomal proteasome. At 10 μM, none of the compounds affected the proteasomal trypsin-like activity in cell lysates of bloodstream forms of T. brucei. On the other hand, leupeptin, a well-established β2 inhibitor, supressed the proteasomal trypsin-like activity within trypanosome cell lysates with a 50% inhibitory concentration of 2 μM demonstrating the inhibitability of the trypsin-like activity of the T. brucei proteasome under the experimental condition. Nevertheless, two compounds (LU-102 and LU-002i) displayed moderate trypanocidal activity with 50% growth inhibition values of 6.9 and 8.5 μM, respectively. In the case of LU-102, it was shown that the trypanocidal activity of the compound was due to inhibition of the major lysosomal cysteine protease Tb CATL. The main finding of this study indicate substantial inhibitor sensitivity differences between the trypsin-like sites of the human and trypanosomal proteasomes. Whether these differences can be exploited for the design of anti-trypanosomal drug therapies remains to be shown. [ABSTRACT FROM AUTHOR]

Abstract: Transferrin (Tf) is a dumbbell-shaped iron transport protein composed of two homologous lobes (C-lobe and N-lobe) and is an essential growth factor for the protozoan parasite Trypanosoma brucei. The trypanosomal receptor for Tf uptake (TbTfR) is a heterodimeric complex that bears no structural similarity with the human Tf receptor. As a first step in identifying the region of Tf involved in binding to the TbTfR, C-lobe and N-lobe fragments were assessed for their capability to interact with the receptor. Preparations of C-lobe and N-lobe fragments were obtained by digestion of iron-loaded bovine Tf with proteinase K-agarose. The individual fragments were then purified by concanavalin A affinity chromatography. Uptake experiments with bloodstream forms of T. brucei demonstrated that both C-lobe and N-lobe fragments were ingested by the parasites. The uptake of the isolated lobes could be inhibited by an excess of Tf and vice versa. Dot blot binding assays showed that both C-lobe and N-lobe fragments were capable of binding to the TbTfR. Both isolated lobes were also able to support the growth of bloodstream forms of T. brucei when cultured in Tf-depleted medium. However, the C-lobe fragment was more efficiently taken up and more potent in supporting parasite growth. The results indicate that the interaction of Tf with the TbTfR is different from that with the human Tf receptor. This difference may be exploited for the development of agents specifically interfering with the binding of Tf to the TbTfR. [Copyright &y& Elsevier]

Abstract: The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target. [Copyright &y& Elsevier]

Abstract: Cysteine peptidases are important for growth and survival of kinetoplastid parasites. The best characterised are those homologous to mammalian cathepsins B and L. To address a somewhat confusing terminology, we introduce a unifying nomenclature for kinetoplastid CATB and CATL peptidases. We review their evolutionary relatedness, genomic organisation, developmental expression, subcellular location and physiological functions. In addition, the applications of kinetoplastid CATB and CATL enzymes as vaccine candidates, diagnostic markers and drug targets are discussed. [Copyright &y& Elsevier]

The majority of the world''s population still live without access to healthy water and the contamination of drinking water with protozoan pathogens poses a serious threat to millions of people in the developing world. Even in the developed world periodic outbreaks of diarrhoeal diseases are caused by the protozoan parasites Cryptosporidium sp., Giardia duodenalis and Entamoeba histolytica. Thus, surveillance of drinking water is imperative to minimize such contaminations and ensure continuous supplies of healthy water world-wide. This article reviews the progress in technology for detection and surveillance of these important waterborne parasites. [Copyright &y& Elsevier]

Abstract: The transferrin (Tf) receptor of Trypanosoma brucei (TbTfR) is encoded by two expression-site-associated genes, ESAG6 and ESAG7. There are around 20 different expression sites containing different copies of these genes that encode TbTfRs with quite distinct affinities for Tf of various hosts. It was proposed that T. brucei has developed multiple expression sites encoding different TbTfRs to ensure sufficient iron uptake in the presence of antibodies competing for binding to Tf. Here it is shown that anti-TbTfR antibody titres produced during chronic murine trypanosomiasis are only one-tenth of those achieved by immunisation of mice using recombinant TbTfR. Calculations indicate that the concentrations of competing anti-TbTfR antibodies present during chronic T. brucei infection are too low to deprive the parasite of iron. In addition, during human African trypanosomiasis the antibody response to the TbTfR seems to be poor and transient. Altogether, the results suggest that the host antibody response to the TbTfR during chronic infection with T. brucei is too low, if present at all, to prevent sufficient iron uptake by bloodstream forms to promote their growth. [Copyright &y& Elsevier]

• The term 'rhodesain' is tainted due to its association with Cecil Rhodes. • The protein sequence of rhodesain is essentially identical to that of brucipain. • The term Tbr CATL (T. brucei cathepsin L) is proposed instead of rhodesain. Rhodesain is the generic name for the cathepsin L-like peptidase of Trypanosoma brucei rhodesiense. The term rhodesain was derived from the subspecies epithet rhodesiense which itself originated form Rhodesia, a historical region in southern Africa named after the 19th century British imperialist and white supremacist Cecil Rhodes. This tainting could be grounds for discontinuing the name, however, there are also scientific grounds. Specifically, protein sequence comparisons and frequency-based difference profiling reveal that rhodesain is essentially identical (99.87−98.44%) to the cathepsin L-like peptidases of both T. b. brucei and T. b. gambiense. Accordingly, and based on a previously proposed terminology for kinetoplastid C1 peptidases (Caffrey and Steverding, 2009), we suggest the use of the formal term, Tbr CATL, to denote the cathepsin L-like peptidases of the T. brucei subspecies. The earlier and informal term, 'brucipain', could also be used. [ABSTRACT FROM AUTHOR]

• ESAG6 with a transmembrane domain (ESAG6tmd) was inducibly expressed in T. brucei. • ESAG6tmd is less heterogeneously glycosylated than GPI-anchored ESAG6. • ESAG6tmd can dimerise with ESAG7 to form a T. brucei transferrin receptor (Tb TfR). • Transmembrane-anchored Tb TfR is as efficient in Tf uptake as a GPI-anchored Tb TfR. The transferrin receptor of Trypanosoma brucei (Tb TfR) is a heterodimer of a glycosylphosphatidylinositol (GPI)-anchored ESAG6 subunit and an ESAG7 subunit. To investigate whether the GPI-anchor is essential for the function of the Tb TfR, an ESAG6 with a transmembrane domain instead of a GPI-anchor (ESAG6tmd) was inducibly expressed in bloodstream form trypanosomes. It is shown that the ESAG6tmd is able to dimerise with ESAG7 to form a Tb TfR that can bind transferrin. Fractionation experiments clearly demonstrated that the transmembrane-anchored Tb TfR is exclusively associated with the membrane fraction. No difference in the uptake of transferrin was observed between trypanosomes inducibly expressing a transmembrane-anchored Tb TfR and trypanosomes inducibly expressing a GPI-anchored Tb TfR. Differences in glycosylation pattern of ESAG6tmd and native ESAG6 may indicate different intracellular trafficking of transmembrane- and GPI-anchored Tb TfRs. The findings suggest that the GPI-anchor is not essential for the function of the Tb TfR in bloodstream forms of T. brucei. [ABSTRACT FROM AUTHOR]

• The vinyl sulfone LU-102 selectively inhibits Tb CATL without affecting Tb CATB. • LU-102 does not affect the trypsin-like activity of the trypanosomal proteasome. • Inhibition of Tb CATL alone is sufficient to kill T. brucei bloodstream forms. The lysosomal cysteine protease activity of Trypanosoma brucei comprises a cathepsin B enzyme (Tb CATB) and a cathepsin L enzyme (Tb CATL). Inhibition of the cysteine protease activity is lethal to bloodstream-form trypanosomes but it was not entirely clear which of the two enzymes are essential for survival of the parasites. Here we show that the vinyl sulfone compound LU-102 selectively inhibits Tb CATL without affecting Tb CATB and the proteasomal trypsin-like activity within trypanosomes. Therefore, the trypanocidal activity displayed by LU-102 can be attributed solely to the inhibition of Tb CATL demonstrating that this enzyme is essential to the survival of T. brucei. [ABSTRACT FROM AUTHOR]

The transferrin receptor of Trypanosoma brucei is encoded by genes located in different expression sites. The various expression sites encode slightly different transferrin receptors, which differ substantially in their affinity for transferrin of different host species. It was proposed that T. brucei has developed multiple expression sites encoding different transferrin receptors not only to cope with the diversity of mammalian transferrins, but also to ensure sufficient iron uptake in the presence of anti-transferrin receptor antibodies. This article shows that calculations based on Kd values argue against the first part of the hypothesis, but might support the second part. [Copyright &y& Elsevier]

Ivermectin is a sixteen-membered macrolactone "wonder drug" of Nobel prize-honored distinction that exhibits a wide range of antiparasitic activities. It has been used for almost four decades in the treatment of various parasitic diseases in humans and animals. In this paper, we describe the synthesis of the first-in-class ivermectin derivatives obtained via derivatization of the C13 position, along with the unexpected rearrangement of the oxahydrindene (hexahydrobenzofuran) unit of the macrolide ring. The structural investigation of the rearrangement has been performed using the single-crystal X-ray diffraction method. The antiparasitic and cytotoxic activities of the newly synthesized derivatives were determined in vitro with the bloodstream form of Trypanosoma brucei brucei , the hepatic stage of Plasmodium berghei , and human leukemia HL-60 cells. The compounds with the highest trypanocidal activity were the C13- epi -2-chloroacetamide analogs of native (6h) or rearranged (7h) ivermectin. Both 6h and 7h displayed trypanocidal activities within a similar mid-nanomolar concentration range as the commercially used trypanocides suramin and ethidium bromide. Furthermore, 6h and 7h exhibited a comparable cytotoxic to trypanocidal ratio as the reference drug ethidium bromide. The double-modified compound 7a (C13- epi -acetamide of rearranged ivermectin) exhibited the highest activity against P. berghei grown in human hepatoma cells, which was 2.5 times higher than that of ivermectin. The findings of this study suggest that C13- epi -amide derivatives of ivermectin are suitable leads in the rational development of new antiparasitic agents. [Display omitted] • An unusual rearrangement of the oxahydrindene (hexahydrobenzofuran) unit of ivermectin was discovered. • The rearrangement led to new ivermectin derivatives. • Ivermectin derivatives displayed promising antimalarial and antitrypanosomal activities. [ABSTRACT FROM AUTHOR]

Salinomycin (SAL) is a natural polyether ionophore that exhibits a very broad spectrum of biological effects, ranging from anticancer to antiparasitic activities. Our recent studies have shown that the chemical modification of the SAL biomolecule is a fruitful strategy for generating lead compounds for the development of novel antitrypanosomal agents. As a continuation of our program to develop trypanocidal active lead structures, we synthesized a series of 14 novel urea and thiourea analogs of C20- epi -aminosalinomycin (compound 2b). The trypanocidal and cytotoxic activities of the derivatives were assessed with the mammalian life cycle stage of Trypanosoma brucei and human leukemic HL-60 cells, respectively. The most antitrypanosomal compounds were the two thiourea derivatives 4b (C20- n -butylthiourea) and 4d (C20-phenylthiourea) with 50% growth inhibition (GI 50) values of 0.18 and 0.22 μM and selectivity indices of 47 and 41, respectively. As potent SAL derivatives have been shown to induce strong cell swelling in bloodstream forms of T. brucei , the effect of compounds 4b and 4d to increase the cell volume of the parasite was also investigated. Interestingly, both derivatives were capable to induce faster cell swelling in bloodstream-form trypanosomes than the reference compound SAL. These findings support the suggestion that C20- epi -aminosalinomycin derivatives are suitable leads in the rational development of new and improved trypanocidal drugs. [Display omitted] • A series of novel (thio)urea derivatives of salinomycin (SAL) was synthesized. • Six compounds (3c , 3d , and 4a‒d) showed promising antitrypanosomal activities. • Several compounds showed a good selectivity of action. • Compounds 4b and 4d displayed faster cell swelling activity than native SAL. [ABSTRACT FROM AUTHOR]

Abstract: Iron is an essential growth component in all living organisms and plays a central role in numerous biochemical processes due to its redox potential and high affinity for oxygen. The use of iron chelators has been suggested as a novel therapeutic approach towards parasitic infections, such as malaria, sleeping sickness and leishmaniasis. Known iron chelating agents such as Deferoxamine and the 3-hydroxypyridin-4-one (HPO) Deferiprone possess anti-parasitic activity but suffer from mammalian toxicity, relatively modest potency, and/or poor oral availability. In this study, we have developed novel derivatives of Deferiprone with increased anti-parasitic activity and reduced cytotoxicity against human cell lines. Of particular interest are several new derivatives in which the HPO scaffold has been conjugated, via a linker, to the 4-aminoquinoline ring system present in the known anti-malaria drug Chloroquine. We report the inhibitory activity of these novel analogues against four parasitic protozoa, Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum, and, for direct comparison, against human cells lines. We also present data, which support the hypothesis that iron starvation is the major cause of growth inhibition of these new Deferiprone–Chloroquine conjugates in T. brucei. [Copyright &y& Elsevier]

Polyether ionophores, with >120 molecules belonging to this group, represent a class of naturally-occurring compounds that exhibit a broad range of pharmacological properties, including promising activity towards a variety of parasites. In this context, salinomycin (SAL) seems to be interesting, as this ionophore has been found to be active against parasites that are responsible for a number of human and animal diseases. On the other hand, less explored is the investigation into the anti-parasitic activity of SAL derivatives. Recently, we identified C1 amides and esters of SAL and its analogue, C20-oxosalinomycin, as promising structures for trypanocidal drug candidates. In search for novel compounds effective against African trypanosomes, the synthetic access to a completely new series of C20- epi -salinomycin (compound 2) analogues is described in this paper. This series includes products obtained via derivatisation of either the C1 carboxyl or the C20 hydroxyl of 2 , but also C1/C20 double modified derivatives. The anti-trypanosomal activity as well as the cytotoxic activity of these analogues were evaluated with bloodstream forms of T. brucei and human myeloid HL-60 cells, respectively. It was found that the C20 single modified derivatives 8 , 12 , and 18 (C20 decanoate, C20 ethyl carbonate, and C20 allophanate of 2 , respectively) were the most active compounds in selectively targeting bloodstream-form trypanosomes, with 50% growth inhibition (GI 50) values of 0.027–0.043 μM and selectivity indices of 165–353. These results indicate that modification at the C20 position of C20- epi -salinomycin 2 can provide semi-synthetic products with enhanced trypanocidal activity that could be of great value for the development of new drugs to treat African trypanosomiasis. Image 1 • A series of new analogues of salinomycin (SAL) was synthesized. • Three novel analogues 8 , 12 and 18 showed promising trypanocidal activities. • Several derivatives were highly selective compounds. • Six analogues met the activity criteria for hit compounds for T. brucei. • Mechanism of action of trypanocidal derivatives was due to enhanced cell swelling. [ABSTRACT FROM AUTHOR]

Parasitic diseases still pose a serious threat to human and animal health, particularly for millions of people and their livelihoods in low-income countries. Therefore, research into the development of effective antiparasitic drugs remains a priority. Ivermectin, a sixteen-membered macrocyclic lactone, exhibits a broad spectrum of antiparasitic activities, which, combined with its low toxicity, has allowed the drug to be widely used in the treatment of parasitic diseases affecting humans and animals. In addition to its licensed use against river blindness and strongyloidiasis in humans, and against roundworm and arthropod infestations in animals, ivermectin is also used "off-label" to treat many other worm-related parasitic diseases, particularly in domestic animals. In addition, several experimental studies indicate that ivermectin displays also potent activity against viruses, bacteria, protozoans, trematodes, and insects. This review article summarizes the last 40 years of research on the antiparasitic effects of ivermectin, and the use of the drug in the treatment of parasitic diseases in humans and animals. [Display omitted] • The licensed use of ivermectin in humans and animals is described. • The "off-label" use of ivermectin against worm-related parasitic diseases is outlined. • The use of ivermectin in experimental therapies is detailed. • The opportunities and challenges of ivermectin are discussed. [ABSTRACT FROM AUTHOR]

[Display omitted] Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure–activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para / para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use. [ABSTRACT FROM AUTHOR]

[Display omitted] The synthesis and biological evaluation of eleven derivatives of the natural polyether ionophore monensin A (MON), modified at the C-26 position, is presented. Eight urethane and three ester derivatives were tested for their antimicrobial activity against different strains of Staphylococcus aureus , Staphylococcus epidermidis , Escherichia coli and Pseudomonas aeruginosa. In addition, their antiparasitic activity was also evaluated with bloodstream forms of Trypanosoma brucei. The majority of the modified ionophores were active against a variety of Gram-positive bacterial strains, including methicillin-resistant S. epidermidis, and showed better antibacterial activity than the unmodified MON. The phenyl urethane derivative of MON exhibited the most promising antibacterial activity of all tested compounds, with minimal inhibitory concentration values of 0.25–0.50 μg/ml. In contrast, none of the MON derivatives displayed higher antitrypanosomal activity than the unmodified ionophore. [ABSTRACT FROM AUTHOR]

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of γ-phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates. [ABSTRACT FROM AUTHOR]

We have developed a real-time PCR assay for detection of Trypanosoma brucei DNA in human blood samples. The PCR was conducted with newly designed primers targeting the 177-bp repeat satellite DNA in T. brucei and with Sybr Green to monitor the amplicon accumulation. DNA purification using Chelex 100® resin was performed on blood samples collected on Whatman FTA® cards and was shown to be a simple and quantitative method as revealed by real-time PCR. The detection limit of the assay was 100 trypanosomes per mL blood, corresponding to an analytical sensitivity of 0.1 genome equivalents. Trypanosome DNA was detected in all blood samples from sleeping sickness patients and, furthermore, the identity of the amplicon was confirmed in all assays by dissociation analysis. Although template DNA from blood samples was amplified with significantly lower efficiency than genomic DNA, similar efficiency between all assays ensured quantitative results. No amplicon product was obtained with samples from uninfected individuals. The results indicate that the real-time PCR assay described is a rapid and sensitive method suitable for the detection of T. brucei in human blood samples in routine clinical laboratory practice. [Copyright &y& Elsevier]

Previous studies have shown that the proteasome of Trypanosoma brucei is a candidate for novel chemotherapy of African sleeping sickness. In this study, two potent and highly selective α′,β′-epoxyketones peptide proteasome inhibitors, epoxomicin and YU101, have been tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of T. brucei. Both inhibitors displayed promising anti-trypanosomal activities with ED50 and ED90 values in the low to mid nanomolar range. Based on MIC values, epoxomicin exhibited a selectivity index approaching those of commercially available drugs. Enzymatic analyses of proteasomal peptidase activities revealed that, compared with mammalian cells, trypanosomes are particular sensitive to inhibition of the trypsin-like activity of the proteasome. In conclusion, the data suggests that proteasome inhibitors targeting the trypsin-like activity are the rational choice for future anti-trypanosomal drug development. [Copyright &y& Elsevier]

The major lysosomal cysteine proteinase of African trypanosomes is a candidate target for novel chemotherapy of sleeping sickness. This cathepsin L-like enzyme is termed rhodesain and brucipain in Trypanosoma brucei rhodesiense and Trypanosoma brucei brucei, respectively. Three potent and selective dipeptidyl cathepsin L inhibitors have been investigated for their trypanocidal activities in vitro using culture-adapted bloodstream forms of T. b. brucei. Compared with general cysteine proteinase inhibitors used previously by ourselves and others, the present inhibitors had improved selectivity indices and, importantly, anti-trypanosomal activities comparable with those of commercial anti-sleeping sickness drugs. Using purified recombinant rhodesain, potent kinact/Ki values of up to 2.3×106 M−1 s−1 were recorded with the inhibitors. Also, all inhibitors blocked proteinolysis in the lysosome consistent with the inhibition of rhodesain/brucipain. In conclusion, the data support the potential of cathepsin L inhibitors for rational anti-trypanosomal drug development. [Copyright &y& Elsevier]

Treatment of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine inhibits the proliferation of the parasites. Compared with mammalian cells, bloodstream forms of Trypanosoma brucei are 10 times more sensitive to iron depletion. The primary target of the chelator is obviously the intracellular iron as the toxicity of deferoxamine is abolished by addition of holotransferrin, the exogenous source of iron for the parasite. To identify probable target sites, the effect of deferoxamine on ribonucleotide reductase, alternative oxidase and superoxide dismutase, three iron-dependent enzymes in bloodstream-form trypanosomes, was studied. Incubation of the parasites with the chelator leads to inhibition of DNA synthesis and lowers oxygen consumption indicating that deferoxamine may affect ribonucleotide reductase and alternative oxidase. The compound does not inhibit the holoenzymes directly but probably acts by chelating cellular iron thus preventing its incorporation into the newly synthesised apoproteins. Treatment of the parasites with deferoxamine for 24 h has no effect on the activity of superoxide dismutase. The results have implications for antitrypanosomal drug development based on specific intervention with the parasite''s iron metabolism. [Copyright &y& Elsevier]

The major cysteine proteinase (brucipain) of Trypanosoma brucei is a target for chemotherapy of African Sleeping Sickness. We have screened a non-peptidyl acyl hydrazide proteinase inhibitor library of 500 compounds for inhibition of brucipain. Those 21 compounds with IC50 values of <40 μM were tested for efficacy against bloodstream forms of T. brucei in cell culture. Eight acyl hydrazides showed 50% or more inhibition of trypanosome replication at <1 μM. The trypanocidal acitivity of the most effective compounds was comparable with those of the commercial antitrypanosomal drugs suramin and diminazene aceturate. However, these acyl hydrazides exhibited varying cytotoxicity towards human HL-60 cells and therefore, only less favourable selectivity indices compared with the commercially available drugs. Nevertheless, the data support the potential of acyl hydrazides as antitrypanosomal chemotherapeutic agents for treatment of sleeping sickness. [Copyright &y& Elsevier]