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.

Luminescent multiplex viability assay for Trypanosoma brucei gambiense

“…Whole cell in vitro high-throughput screenings (HTS) are now in use to discover novel trypanotoxic compounds. However, these HTS assays are almost exclusively performed with one particular non human pathogenic strain: T. b. brucei strain 427 [5-14]. Less often a hit is confirmed in vitro and in vivo on a collection of Trypanozoon strains, including T.b.…”

Discovery of Inhibitors of Trypanosoma brucei by Phenotypic Screening of a Focused Protein Kinase Library

“…Therefore compounds that only exert a static effect (i.e., stopping the growth of cells, whilst not killing them) or a decrease in growth rate are unlikely to clear the parasites completely and should be removed from drug discovery pipelines as early as possible. To provide information regarding the mode of action of the compounds discussed, we selected a number of examples to run in a static–cidal assay . Compounds with either an imidazo[4,5‐ b ]pyrazin‐2(3 H )‐one ( 24, 26, 28, 30 , and 39 ) or imidazo[4,5‐ b ]pyridin‐2(3 H )‐one ( 51 , 55 , and 56 ) core showed growth‐slowing or static behaviour at all concentrations tested, whereas compounds containing a 1 H ‐pyrazolo[3,4‐ b ]pyridine core ( 60 and 63 ) showed cidal behaviour at a minimum cidal concentration of 17 μ m (Table ).…”

“…[b] Compounds were also tested in the static–cidal assay carried out in triplicate to determine minimal cidal concentration (MCC) and static cidal EC 50 values for the first [SC EC 50 (1)] and second [SC EC 50 (2)] sites. EC 50 and SC EC 50 (1) are expected to represent the same mode of action of the compounds and are therefore very similar . [c] GS: growth‐slowing, S: static, C: cidal.…”

“…Determination of cidality against T. brucei bloodstream‐stage cells was performed using a static–cidal (SC) assay as previously reported . Briefly, bloodstream‐form trypanosomes were seeded into 384‐well plates at 4×10 5 mL −1 (50 μL per well), followed by immediate addition of resazurin (50 μ m final) to one of the plates, and all plates were incubated at 37 °C, 5 % CO 2 .…”

Antiparasitic Chemotherapy: From Genomes to Mechanisms

“…In some cases, drugs that target in vivo virulence mechanisms could co-opt the immune mechanisms of the host in clearing parasites. The development of assays that focus on distinct stages of the malaria life-cycle, improvements in growth assays (147) and the development of in vivo models that allow accurate quantification or parasite burden, and that reflect the second CNS-stage of HAT, will all be important here.…”