Kinetoplastida: new therapeutic strategies

New formulations and therapeutic switching of the established drugs, amphotericin B and paromomycin, together with the discovery of miltefosine, have significantly improved the opportunities for treatment of visceral leishmaniasis (VL) chemotherapy. However, for human African trypanosomiasis (HAT), Chagas disease and cutaneous leishmaniases there has been limited progress. For HAT, a novel diamidine, parfuramidine, is in phase III clinical trial for early-stage disease, but for the treatment of late-stage disease there are no new drugs and combinations of eflornithine with melarsoprol or nifurtimox have been the focus of clinical studies. For Chagas disease, different classes of compounds that have validated biochemical targets, sterol biosynthesis methylases and cysteine proteases, are in various stages of development. The genome sequences that are now available for the pathogens that cause the leishmaniases and trypanosomiases, and new methods for rapid validation of targets, are part of the solution to discover new drugs. The integration of medicinal chemistry, pharmacokinetics, project planning and interaction with the pharma/biotech sector are essential if progress is to be made. Although there are financial constraints, the appearance of new funding sources and not-for-profit product development partnerships offers hope for drug development.     

Chemotherapy of trypanosomiases and leishmaniasis

New formulations, therapeutic switching of the established drugs amphotericin B and paromomycin, and the serendipitous discovery of miltefosine have markedly improved leishmaniasis chemotherapy in the past 21 years. The situation for the two trypanosomiases has been less encouraging. Apart from the introduction of eflornithine for the treatment of late-stage human African trypanosomiasis, with its serious limitations in terms of cost and difficulty of administration, no new drugs have been incorporated into the chemotherapeutic arsenal in the past 25 years, despite important advances in knowledge of the biology of the etiological agents and the pathophysiology of these diseases. In the case of Chagas disease, several classes of compound that target the validated biochemical pathways of the parasite (e.g. inhibitors of sterol biosynthesis and cysteine proteases) are in the pipeline. With the availability of complete genome sequences for all three pathogens, and methods for rapid validation of targets, it is hoped that much-needed amelioration will occur soon. Financial constraints continue to represent a major hurdle to drug development. However, the appearance of not-for-profit product-development partnerships offers a new paradigm for bringing new drugs to patients.     

Trypanosomiasis control

In July 2000, Heads of State of the 36th Session of the Organisation for African Unity signed a potentially important declaration on African trypanosomiasis, urging member states "to act collectively to rise to the challenge of eliminating the problem through concerted efforts in mobilising the necessary human, financial and material resources required to render Africa tsetse-free within the shortest time possible". To many, such an ambitious dream is received with some scepticism, recalling the doubts that surrounded a similar declaration signed in Brasilia in 1991, which paved the way for the Southern Cone Initiative against American trypanosomiasis (Chagas disease). True, the two diseases are quite different. But the operational challenges are quite similar, and there are sufficient biological parallels to suggest that the Latin American experience in controlling Chagas disease may provide a useful model for the control of African trypanosomiasis.     

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone,a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 M, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 M, indicating a good selectivity against the parasite enzyme.     

SAR studies on azasterols as potential anti-trypanosomal and anti-leishmanial agents

There is an urgent need for the development of new drugs for the treatment of neglected tropical diseases such as human African trypanosomiasis, Chagas disease and leishmaniasis. Azasterols, have been shown to have activity against the parasites which cause these diseases. In this paper we report synthesis of new azasterols and subsequent analysis of the SAR. The chemistry focused on variations in the ester at the 3β-position of the sterol and the position of the nitrogen in the side chain. The data allowed us to derive preliminary pharmacophore models for the activity of the azasterols against the parasites which cause these diseases.     

In vitro trypanocidal activity of the anti-helminthic drug niclosamide

Only a few drugs are available for chemotherapy of African trypanosomiasis and there is an urgent need for the development of new anti-trypanosomal agents. In this study, the anti-helminthic drug niclosamide was tested for its trypanocidal activity in vitro using culture-adapted bloodstream forms of Trypanosoma brucei brucei and Trypanosoma congolense. The concentrations of niclosamide to reduce the growth rate by 50% and to kill all cells were in the low- and mid micromolar ranges for T. b. brucei and T. congolense, respectively. The very low toxicity of niclosamide for mammals makes the compound interesting for drug development for African trypanosomiasis.     

Biological evaluation of glycosyl-isoindigo derivatives against the pathogenic agents of tropical diseases (malaria, Chagas disease, leishmaniasis and human African trypanosomiasis)

The biological activities of diversely substituted glycosyl-isoindigo derivatives against the causative agents of tropical diseases (malaria, Chagas disease, leishmaniasis and human African trypanosomiasis) are reported. Some of the compounds tested showed interesting activities with good selectivity indices, particularly against Trypanosoma brucei rhodesiense. These results suggested, for the first time, that glycosyl-isoindigo derivatives could be of interest for the discovery of new lead compounds to treat tropical diseases.     

African trypanosomiasis research: 100 years of progress, but questions and problems still remain

Past and present progress in our understanding of African trypanosomiasis is briefly reviewed. Although tremendous scientific strides have been achieved, an epidemic of the disease is currently underway. Three areas of research which are believed necessary for the control of African trypanosomiasis are discussed. It is suggested that a better understanding of the host-parasite relationship is essential; more emphasis and a broader approach to drug development is required; and finally, further research into the socio-economic aspects of African trypanosomiasis is urgently needed before the human disease can again be controlled.     

Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.     

Preparation of transition-state analogues of sterol 24-methyl transferase as potential anti-parasitics

There is an urgent need for new drugs to treat leishmaniasis and Chagas disease. One important drug target in these organisms is sterol biosynthesis. In these organisms the main endogenous sterols are ergosta- and stigmata-like compounds in contrast to the situation in mammals, which have cholesterol as the sole sterol. In this paper we discuss the design, synthesis and evaluation of potential transition state analogues of the enzyme Delta24(25)-methyltransferase (24-SMT). This enzyme is essential for the biosynthesis of ergosterol, but not required for the biosynthesis of cholesterol. A series of compounds were successfully synthesised in which mimics of the S-adenosyl methionine co-factor were attached to the sterol nucleus. Compounds were evaluated against recombinant Leishmania major 24-SMT and the parasites L. donovani and Trypanosoma cruzi in vitro, causative organisms of leishmaniasis and Chagas disease, respectively. Some of the compounds showed inhibition of the recombinant Leishmania major 24-SMT and induced growth inhibition of the parasites. Some compounds also showed anti-parasitic activity against L. donovani and T. cruzi, but no inhibition of the enzyme. In addition, some of the compounds had anti-proliferative activity against the bloodstream forms of Trypanosoma brucei rhodesiense, which causes African trypanosomiasis.     

Modelling the impact of fexinidazole use on human African trypanosomiasis (HAT) transmission in the Democratic Republic of the Congo

Gambiense human African trypanosomiasis is a deadly disease that has been declining in incidence since the start of the Century, primarily due to increased screening, diagnosis and treatment of infected people. The main treatment regimen currently in use requires a lumbar puncture as part of the diagnostic process to determine disease stage and hospital admission for drug administration. Fexinidazole is a new oral treatment for stage 1 and non-severe stage 2 human African trypanosomiasis. The World Health Organization has recently incorporated fexinidazole into its treatment guidelines for human African trypanosomiasis. The treatment does not require hospital admission or a lumbar puncture for all patients, which is likely to ease access for patients; however, it does require concomitant food intake, which is likely to reduce adherence. Here, we use a mathematical model calibrated to case and screening data from Mushie territory, in the Democratic Republic of the Congo, to explore the potential negative impact of poor compliance to an oral treatment, and potential gains to be made from increases in the rate at which patients seek treatment. We find that reductions in compliance in treatment of stage 1 cases are projected to result in the largest increase in further transmission of the disease, with failing to cure stage 2 cases also posing a smaller concern. Reductions in compliance may be offset by increases in the rate at which cases are passively detected. Efforts should therefore be made to ensure good adherence for stage 1 patients to treatment with fexinidazole and to improve access to care.     

Trypanosoma cruzi populations: more clonal than sexual

The ancient question of trypanosome sexuality has recently been reactivated in view of important observations in the African species Trypanosoma brucie, in which Mendelian sexuality has been proposed as a working hypothesis on the basis o f indirect isozyme evidence. Subsequent experiments have confirmed that recombination can occur in T. brucei under defined experimental conditions and suggest that this parasite undergoes meiosis. In this article, Michel Tibayrenc and Francisco Ayala discuss the intraspecific variability of another species, Tyapanosoma cruzi - causative agent of american trypanosomiasis or Chagas disease. They interpret the variation revealed by extensive isozyme analysis and restriction endonuclease analysis of kinetoplast DNA, to suggest that T. cruzi is diploid, genetically very polymorphic, and has a clonal structure that manifests a lack of (or very restricted) sexuality.     

Nifurtimox activation by trypanosomal type I nitroreductases generates cytotoxic nitrile metabolites

The prodrug nifurtimox has been used for more than 40 years to treat Chagas disease and forms part of a recently approved combinational therapy that targets West African trypanosomiasis. Despite this, its mode of action is poorly understood. Detection of reactive oxygen and nitrogen intermediates in nifurtimox-treated extracts led to the proposal that this drug induces oxidative stress in the target cell. Here, we outline an alternative mechanism involving reductive activation by a eukaryotic type I nitroreductase. Several enzymes proposed to metabolize nifurtimox, including prostaglandin F2α synthase and cytochrome P450 reductase, were overexpressed in bloodstream-form Trypanosoma brucei. Only cells with elevated levels of the nitroreductase displayed altered susceptibility to this nitrofuran, implying a key role in drug action. Reduction of nifurtimox by this enzyme was shown to be insensitive to oxygen and yields a product characterized by LC/MS as an unsaturated open-chain nitrile. This metabolite was shown to inhibit both parasite and mammalian cell growth at equivalent concentrations, in marked contrast to the parental prodrug. These experiments indicate that the basis for the selectivity of nifurtimox against T. brucei lies in the expression of a parasite-encoded type I nitroreductase.     

Squalamine analogues as potential anti-trypanosomal and anti-leishmanial compounds

This paper concerns the synthesis of various simplified analogues of the novel anti-microbial agent, squalamine. The compounds were then investigated for activity against Trypanosoma brucei, the causative agent of African trypanosomiasis, Trypanosoma cruzi, the causative agent of Chagas disease and Leishmania donovani, the causative agent of visceral leishmaniasis. Several compounds showed in vitro activity, especially against T. brucei and L. donovani. However, one compound showed poor in vivo activity.     

Therapy of human African trypanosomiasis: current situation

This paper is a review of the current situation of the treatment of human African trypanosomiasis. The existing approved drugs are old, toxic and/or expensive. Therapeutic failures are common. Several factors may contribute to the problems of chemotherapy, including differences in the epidemiology of the disease, difficulties in the diagnosis and staging of the infection, availability, distribution and pharmacologic properties of drugs, standardization of treatment regimens, response to therapy, follow-up period, and relapses and clinical trials. The new therapeutic approaches include the development and approval of new drugs, the use of new therapeutic regimens, the study of drug combinations, and the development of new formulations.     

Waking up to sleeping sickness

Devastating epidemics of human African trypanosomiasis are currently re-emerging in many sub-Saharan countries. In the past three decades, clinical research into this important disease has been neglected, as have urgently needed initiatives on drug development, disease surveillance and vector control. Recent impetus has aimed to provide a free supply of antitrypanosomal drugs, to develop a new orally active trypanocidal agent and to attack the tsetse vector with modern technology. In addition, pan-African initiatives to co-ordinate control efforts have begun. These all provide some hope for the future, but they might not be enough to reverse the resurgence of this deadly disease in the heart of Africa.     

Nanotechnology based drug delivery system(s) for the management of tuberculosis

The era of nanotechnology has allowed new research strategies to flourish in the field of drug delivery. Nanoparticle-based drug delivery systems are suitable for targeting chronic intracellular infections such as tuberculosis. Polymeric nanoparticles employing poly lactide-co-glycolide have shown promise as far as intermittent chemotherapy in experimental tuberculosis is concerned. It has distinct advantages over the more traditional drug carriers, i.e. liposomes and microparticles. Although the experience with natural carriers, e.g. solid lipid nanoparticles and alginate nanoparticles is in its infancy, future research may rely heavily on these carrier systems. Given the options for oral as well as parenteral therapy, the very nature of the disease and its complex treatment urges one to emphasize on the oral route for controlled drug delivery. Pending the discovery of more potent antitubercular drugs, nanotechnology-based intermittent chemotherapy provides a novel and sound platform for an onslaught against tuberculosis.     

Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor

High systemic drug toxicity and increasing prevalence of drug resistance hampers efficient treatment of human African trypanosomiasis (HAT). Hence, development of new highly specific trypanocidal drugs is necessary. Normal human serum (NHS) contains apolipoprotein L-I (apoL-I), which lyses African trypanosomes except resistant forms such as Trypanosoma brucei rhodesiense. T. b. rhodesiense expresses the apoL-I-neutralizing serum resistance-associated (SRA) protein, endowing this parasite with the ability to infect humans and cause HAT. A truncated apoL-I (Tr-apoL-I) has been engineered by deleting its SRA-interacting domain, which makes it lytic for T. b. rhodesiense. Here, we conjugated Tr-apoL-I with a single-domain antibody (nanobody) that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of trypanosomes to generate a new manmade type of immunotoxin with potential for trypanosomiasis therapy. Treatment with this engineered conjugate resulted in clear curative and alleviating effects on acute and chronic infections of mice with both NHS-resistant and NHS-sensitive trypanosomes.     

The Trypanosoma cruzi virulence factor oligopeptidase B (OPBTc) assembles into an active and stable dimer

Oligopeptidase B, a processing enzyme of the prolyl oligopeptidase family, is considered as an important virulence factor in trypanosomiasis. Trypanosoma cruzi oligopeptidase B (OPBTc) is involved in host cell invasion by generating a Ca(2+)-agonist necessary for recruitment and fusion of host lysosomes at the site of parasite attachment. The underlying mechanism remains unknown and further structural and functional characterization of OPBTc may help clarify its physiological function and lead to the development of new therapeutic molecules to treat Chagas disease. In the present work, size exclusion chromatography and analytical ultracentrifugation experiments demonstrate that OPBTc is a dimer in solution, an association salt and pH-resistant and independent of intermolecular disulfide bonds. The enzyme retains its dimeric structure and is fully active up to 42°C. OPBTc is inactivated and its tertiary, but not secondary, structure is disrupted at higher temperatures, as monitored by circular dichroism and fluorescence spectroscopy. It has a highly stable secondary structure over a broad range of pH, undergoes subtle tertiary structure changes at low pH and is less stable under moderate ionic strength conditions. These results bring new insights into the structural properties of OPBTc, contributing to future studies on the rational design of OPBTc inhibitors as a promising strategy for Chagas disease chemotherapy.     

An overview of Chagas disease treatment

Chagas disease (American trypanosomiasis) is endemic in 21 countries of the Americas, where control is largely focused on elimination of the domestic insect vectors (Triatominae) coupled with measures to extend and improve the screening of blood donors in order to avoid tranfusional transmission. Through national programmes and multinational initiatives coordinated by WHO-PAHO, much has been accomplished in these domains in terms of reducing transmission. Attention now turns to consolidating the successes in interrupting transmission, and improved treatment for those already infected and those who may become affected in the future. This article, based on technical discussions at the "Epidemiological and Sociological Determinants of Chagas Disease, Basic Information to Establish a Surveillance and Control Policy " meeting in Rio de Janeiro, is designed to open the debate on appropriate strategies for continuation of the successful initiatives against Chagas disease.