Category: Assay Development and Screening
The Division of Experimental Therapeutics (ET) at Walter Reed Army Institute of Research is engaged in antiparasitic drug development. The foundation of that effort rests on in vitro screening to find potent anti-malarial and anti-leishmanial compounds as prospective drugs to treat United States Army soldiers. We have significantly increased our ability to advance potential antiparasitic therapeutics to the next steps in the drug discovery pipeline through automation of all our antiparasitic in vitro potency, toxicity, and pharmacokinetics screens. We utilize three in vitro assays for malaria drug discovery: the Malaria SYBR Green I - based Fluorescence Assay (MSF Assay), the Inhibition of Liver Stage Development Assay (ILSDA), and the Malaria Stage 5 Gametocyte Assay. The MSF Assay utilizes SYBR Green, a DNA-intercalating dye, to measure parasitic growth of asexual parasites when treated with possible blood-stage anti-malarial compounds. The ILSDA assay measures the proliferation of luciferase-expressing P. berghei parasites that have invaded HepG2 liver cells in the presence of potential malaria liver-stage active compounds. The Gametocyte assay relies on luciferase-expressing malaria parasites to measure parasite growth when treated with possible anti-malarial compounds that target the sexual stages of malaria. Drug discovery of anti-leishmanial compounds is conducted using an in vitro intracellular amastigote assay that measures the growth of luciferase-expressing leishmania amastigotes developing inside macrophages in the presence of promising anti-leishmanial compounds. All potent anti-malarial and anti-leishmanial compounds must be tested for toxicity, and we use a MTT toxicity assay to assess compound toxicity against HepG2 liver cells. The MTT assay measures the activity of mitochondrial reductases to metabolize a substrate, MTT or 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and cellular respiration of this substrate in the presence of possibly toxic compounds is used as a toxicity endpoint. All compounds that meet the potency and toxicity thresholds of our gated-tier paradigm for antiparasitic drug development are progressed to ET's Drug Metabolism and Disposition Laboratory for in vitro evaluation of pharmacokinetic properties. The DMD Lab conducts solubility, metabolism, and enzyme inhibition studies in order to further triage compounds for advancement. All of our assays were previously performed by hand in a 96-well format, and have since been optimized on robotic platforms in 96- or 384-well format. Cell-based assays have unique liquid handling requirements, which must be met to ensure assay success. Consequently, the optimization and qualification of these assays has involved extensive testing of liquid class variables, such as speed and location of liquid dispensation within the well. The automation and micronization of our in vitro assays has more than doubled our throughput, and will make a considerable impact on antiparasitic drug discovery efforts in support of the United States Army.
Brittney Potter– Laboratory Manager / Bioanalytical Scientist, Walter Reed Army Institute of Research, Silver Spring, MD