Discovery of Trypanocidal Compounds by Whole Cell HTS of Trypanosoma brucei

Abstract

Chemotherapy against human African trypanosomiasis relies on four drugs that cause frequent and occasionally severe side-effects. Because human African trypanosomiasis is a disease of poor people in Africa, the traditional market-driven pathways to drug development are not available. One potentially rapid and cost-effective approach to identifying and developing new trypanocidal drugs would be high throughput-screening of existing drugs already approved for other uses, as well as clinical candidates in late development. We have developed an ATP-bioluminescence assay that could be used to rapidly and efficiently screen compound libraries against trypanosomes in a high throughput-screening format to validate this notion. We screened a collection of 2160 FDA-approved drugs, bioactive compounds and natural products to identify hits that were cytotoxic to cultured Trypanosoma brucei at a concentration of 1 mum or less. This meant that any hit identified would be effective at a concentration readily achievable by standard drug dosing in humans. From the screen, 35 hits from seven different drug categories were identified. These included the two approved trypanocidal drugs, suramin and pentamidine, several other drugs suspected but never validated as trypanocidal, and 17 novel trypanocidal drugs.

Development of an Alamar Blue™ Viability Assay in 384-Well Format for High Throughput Whole Cell Screening of Trypanosoma brucei brucei Bloodstream Form Strain 427

“…For example, an ATP detection assay that has been used in a 96-well format by Mackey and others, 6 when converted to a 384-well format and assuming a volume of 25 μL/well as suggested by the manufacturer, would result in a reagent cost per well 3.8× greater than that given in this report. Although the HTS Alamar Blue assay we report has a total incubation time of 96 hours, batches of plates could potentially be run in relatively large numbers, which is based on a standard incubator capacity that would be approximately 80-100 × 384-well plates in a 5-day cycle.…”

“…A luciferase viability assay adapted to a 96-well format has most recently been used for HTS of a small compound library against Trypanosoma brucei brucei . 6 However, this 96-well method is not suitable for undertaking HTS of larger libraries because of the intensive labor requirements and expense. HTS of known HAT targets, such as trypanothione reductase 7 in a 384-well format are documented, but there are no reports of 384-well assays against the whole parasite.…”

High Throughput Screening against the Peroxidase Cascade of African Trypanosomes Identifies Antiparasitic Compounds That Inactivate Tryparedoxin

“…In a second approach, a luciferase-based viability assay was used. T. brucei and HeLa cells (31) were incubated with the compounds for 72 h, and the cellular ATP level was measured (37). The antitrypanosomal effect of the compounds became apparent after 24 h and remained practically constant for 72 h. The mammalian HeLa cells revealed generally higher EC 50 values, which resulted in selectivity indices of up to Ͼ83 (Table 1).…”

Identification of New Antileishmanial Leads from Hits Obtained by High-Throughput Screening

“…Cell-based phenotypic screening has been useful in identifying new chemotypes against pathogenic protozoa (7,8,11,28). Sharlow et al reported the screening of 196,146 publicly available compounds for activity against Leishmania major promastigotes and identified 17,629 compounds as primary hits (27).…”