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

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. © 2012 de rycker et al.