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      A Static-Cidal Assay for Trypanosoma brucei to Aid Hit Prioritisation for Progression into Drug Discovery Programmes

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          Abstract

          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.

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          Most cited references18

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          The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro.

          Alamar Blue, an indicator for metabolic cell function, was evaluated as a fluorescent and as a colorimetric dye in drug sensitivity assays for human pathogenic African trypanosomes, Trypanosoma brucei rhodesiense and T.b. gambiense. The experimental conditions were adjusted to find those where the relationship between trypanosome number and Alamar Blue signal was linear over the widest possible range. Fluorescent signals correlated to trypanosome numbers from 10(4) trypanosomes/ml (T.b. rhodesiense) and 10(5) trypanosomes/ml (T.b. gambiense) up to 2-3 x 10(6) trypanosomes/ml when trypanosomes were incubated for 2 h with 10% Alamar Blue. Trypanocidal activity of common drugs (melarsoprol, DFMO, suramin, pentamidine and diminazene aceturate) was determined employing this assay. The IC50 values obtained were comparable to those obtained with another fluorochrome, BCECF-AM. The Alamar Blue assay can be applied for drug screening, since it is simple, reproducible and economical. The assay can also be used in field sites with less equipped laboratories, because in addition to fluorometric endpoint determination, a colorimetric reading is possible.
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            N-Myristoyltransferase inhibitors as new leads to treat sleeping sickness

            African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.
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              Polyamine metabolism: a potential therapeutic target in trypanosomes.

              alpha-Difluoromethylornithine (RMI 71,782), a specific irreversible inhibitor of the first step in polyamine biosynthesis, that is, the formation of putrescine from ornithine by ornithine decarboxylase, cures mice infected with a virulent, rodent-passaged strain of Trypanosoma brucei brucei. This parasite is closely related to the trypanosomes that cause human sleeping sickness. The drug, which is remarkably nontoxic, was effective when administered in drinking water or by intubation. The ability of the compound to inhibit ornithine decarboxylase in vitro was demonstrated by the reduced amounts of putrescine synthesized from tritiated ornithine in Trypanosoma brucei suspensions. These observations direct attention to polyamine metabolism as a target for chemotherapy of parasitic diseases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                November 2012
                29 November 2012
                : 6
                : 11
                : e1932
                Affiliations
                [1]Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom
                University of Pittsburgh, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MDR DWG AHF. Performed the experiments: MDR SON DJ LC. Analyzed the data: MDR SON DJ LC DWG. Wrote the paper: MDR AHF.

                [¤]

                Current address: Advinus Therapeutics Ltd., Pune, India

                Article
                PNTD-D-12-00606
                10.1371/journal.pntd.0001932
                3510075
                23209868
                d8f17520-9466-495d-b72a-d1e7e8f8cf56
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 24 May 2012
                : 17 October 2012
                Page count
                Pages: 7
                Funding
                This work was supported by grants from the Wellcome Trust (077705, 079838 and 083481). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biotechnology
                Drug Discovery
                Microbiology
                Parasitology
                Medicine
                Infectious Diseases
                Neglected Tropical Diseases
                African Trypanosomiasis

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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