title: A static-cidal assay for Trypanosoma brucei to aid hit prioritisation for progression into drug discovery programmes.
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.
authors.affiliation: Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdom. M.DeRycker@dundee.ac.uk
authors.forename: Manu
authors.initials: M
authors.lastname: De Rycker
authors.forename: Sandra
authors.initials: S
authors.lastname: O'Neill
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Joshi
authors.forename: Lorna
authors.initials: L
authors.lastname: Campbell
authors.forename: David W
authors.initials: DW
authors.lastname: Gray
authors.forename: Alan H
authors.initials: AH
authors.lastname: Fairlamb
mesh_terms: D017954:Africa South of the Sahara; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D014344:Trypanocidal Agents / Q000302:isolation & purification*; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000254:growth & development
chemical_list: D014344:Trypanocidal Agents