High-Throughput Screening Assay In 384-Well Format For The Identification Of Anti-Trypanosomal Agents Against Trypanosoma Brucei Rhodesiense And Mode Of Cell Death Study
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Date
2017-02
Authors
Lim, Kah Tee
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
In order to accelerate the discovery of novel leads for the treatment of Human
African Trypanosomiasis (HAT), it is necessary to have a simple and sensitive assay
to identify positive hits by whole cell viability based high-throughput screening
(HTS). In this study, the HTS assay was developed in 384-well format using clear
plate and black plate, for comparison. Assay robustness and reproducibility were
determined under the optimized conditions in 384-well plate was well tolerated in the
HTS assay, including percentage of coefficient of variation of 4.68% and 4.74%,
signal-to-background ratio of 12.75 and 12.07, and Z’ factor of 0.79 and 0.82 in
clear- and black-384-well plate, respectively. Final concentration of 0.30% dimethyl
sulfoxide was well tolerated in the HTS assay in both types of plate. No significant
differences were observed for reference drugs pentamidine and melarsoprol used in
the two plate format. Therefore, clear 384-well plate was suitable for use in the HTS
campaign for the identification of new compounds against Trypanosoma brucei
rhodesiense. A total of 1,333 plants collected from Malaysian forest were tested
using the HTS system developed and six plants were found to have potent antitrypanosomal
activity. These plants extract have an IC50 value less than 1.56 μg/mL
with associated selectivity indices (SI) greater than 100. Further testing against T. b.
rhodesiense led to the selection of Senna spectabilis (IP 117; local name: “Kasia
Kuning”) for further bioassay-guided isolation against the causative species of HAT.
Description
Keywords
High-throughput screening assay in 384-well format , identification of anti-trypanosomal agents