Abstract
Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC50 value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC50 values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.
Highlights
Human African Trypanosomiasis (HAT) is caused by infection with either the trypanosome subspecies Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense
Human African Sleeping Sickness (HAT) is a disease caused by sub-species of Trypanosoma
Priority compounds were tested against a panel of protozoan parasites, including Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum
Summary
Human African Trypanosomiasis (HAT) is caused by infection with either the trypanosome subspecies Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. Decreasing numbers of reported new cases over the last 10 years have been reported - from over 25,000 in 2000 to 10,000 in 2009 - of which over 95% are caused by T.b. gambiense [1]. The World Health Organization (WHO) currently estimates the actual number of cases to be around 30,000 [http://www.who.int/mediacentre/factsheets/ fs259/en/]. HAT occurs in two stages, whereby the first stage, called the haemolymphatic stage, corresponds to the invasion of lymph, blood and other tissues by the trypanosomes, and the second stage is associated with parasites crossing the blood-brain barrier and invading the central nervous system (CNS). Symptoms of the second stage of the disease include mental impairment, severe headaches, fever, chronic encephalopathy and an eventual, terminal somnolent state, if the disease remains untreated
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