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Human African Trypanosomiasis is a vector-borne disease of sub-Saharan Africa that causes significant morbidity and mortality. Current therapies have many drawbacks, and there is an urgent need for new, better medicines. Ideally such new treatments should be fast-acting cidal agents that cure the disease in as few doses as possible. Screening assays used for hit-discovery campaigns often do not distinguish cytocidal from cytostatic compounds and further detailed follow-up experiments are required. Such studies usually do not have the throughput required to test the large numbers of hits produced in a primary high-throughput screen. Here, we present a 384-well assay that is compatible with high-throughput screening and provides an initial indication of the cidal nature of a compound. The assay produces growth curves at ten compound concentrations by assessing trypanosome counts at 4, 24 and 48 hours after compound addition. A reduction in trypanosome counts over time is used as a marker for cidal activity. The lowest concentration at which cell killing is seen is a quantitative measure for the cidal activity of the compound. We show that the assay can identify compounds that have trypanostatic activity rather than cidal activity, and importantly, that results from primary high-throughput assays can overestimate the potency of compounds significantly. This is due to biphasic growth inhibition, which remains hidden at low starting cell densities and is revealed in our static-cidal assay. The assay presented here provides an important tool to follow-up hits from high-throughput screening campaigns and avoid progression of compounds that have poor prospects due to lack of cidal activity or overestimated potency. Author Summary: Trypanosoma brucei is a protozoan parasite causing African sleeping sickness. Current treatments for this disease have significant limitations, underlining the need for better and safer drugs. To identify new chemical starting points for drug development, large compound collections are screened against the parasite. Such screens typically do not distinguish between compounds that slow the growth of the parasite and compounds that actually kill the parasite (cidal compounds). Here, we present the development of an assay to identify such compounds. The main advantage of our assay is that it marries a relatively high-throughput to increased understanding of mode of action. Many active compounds (hits) are usually identified in T. brucei primary screening campaigns, making it difficult to select which compounds should undergo further development. Our assay allows testing of all of the hits for cidal activity so that only the most promising compounds are progressed. We show that the starting cell density used in the T. brucei growth assay can have a significant effect on the shape of dose response curves, and that important information regarding the mode of action of a compound can remain hidden at low starting densities as used commonly in T. brucei screening assays. [ABSTRACT FROM AUTHOR]

Human African Trypanosomiasis is a vector-borne disease of sub-Saharan Africa that causes significant morbidity and mortality. Current therapies have many drawbacks, and there is an urgent need for new, better medicines. Ideally such new treatments should be fast-acting cidal agents that cure the disease in as few doses as possible. Screening assays used for hit-discovery campaigns often do not distinguish cytocidal from cytostatic compounds and further detailed follow-up experiments are required. Such studies usually do not have the throughput required to test the large numbers of hits produced in a primary high-throughput screen. Here, we present a 384-well assay that is compatible with high-throughput screening and provides an initial indication of the cidal nature of a compound. The assay produces growth curves at ten compound concentrations by assessing trypanosome counts at 4, 24 and 48 hours after compound addition. A reduction in trypanosome counts over time is used as a marker for cidal activity. The lowest concentration at which cell killing is seen is a quantitative measure for the cidal activity of the compound. We show that the assay can identify compounds that have trypanostatic activity rather than cidal activity, and importantly, that results from primary high-throughput assays can overestimate the potency of compounds significantly. This is due to biphasic growth inhibition, which remains hidden at low starting cell densities and is revealed in our static-cidal assay. The assay presented here provides an important tool to follow-up hits from high-throughput screening campaigns and avoid progression of compounds that have poor prospects due to lack of cidal activity or overestimated potency. [ABSTRACT FROM AUTHOR]

Pyridoxal-5′-phosphate (vitamin B6) is an essential cofactor for many important enzymatic reactions such as transamination and decarboxylation. African trypanosomes are unable to synthesise vitamin B6 de novo and rely on uptake of B6 vitamers such as pyridoxal and pyridoxamine from their hosts, which are subsequently phosphorylated by pyridoxal kinase (PdxK). A conditional null mutant of PdxK was generated in Trypanosoma brucei bloodstream forms showing that this enzyme is essential for growth of the parasite in vitro and for infectivity in mice. Activity of recombinant T. brucei PdxK was comparable to previously published work having a specific activity of 327 ± 13 mU mg−1 and a Kmapp with respect to pyridoxal of 29.6 ± 3.9 µM. A coupled assay was developed demonstrating that the enzyme has equivalent catalytic efficiency with pyridoxal, pyridoxamine and pyridoxine, and that ginkgotoxin is an effective pseudo substrate. A high resolution structure of PdxK in complex with ATP revealed important structural differences with the human enzyme. These findings suggest that pyridoxal kinase is an essential and druggable target that could lead to much needed alternative treatments for this devastating disease. [ABSTRACT FROM AUTHOR]

This article discusses the decline in the effectiveness of antimonial compounds in treating visceral leishmaniasis in Bihar, India. The decrease in efficacy has been attributed to the misuse of antimonials and resistance in parasites. The article proposes an additional hypothesis that chronic exposure to arsenic, which is present in the groundwater in Bihar, may contribute to the decreased efficacy of antimony. The article explores the evidence for this hypothesis and suggests that further research is needed to fully understand the relationship between arsenic contamination and antimonial treatment failure. [Extracted from the article]