At the Swiss Tropical and Public Health Institute, Reto Brun provides a ‘gold standard’ screening centre for protozoan parasites for the World Health Organization (WHO)–Special Programme for Research and Training in Tropical Diseases (TDR) and the Drugs for Neglected Diseases Initiative (DNDi), as well as the Gates-funded Consortium for Parasitic Drug Development (CPDD). His in vitro system involves the cultivation of Trypanosoma cruzi-infected rat myoblasts in 96-well microtitre plates, using genetically modified parasites that express a bacterial enzyme that can be quantified by photometry. This allows the screening of up to 1,000 compounds a week.

At the Sandler Center, husband-and-wife team Juan Engel and Patricia Doyle have created a medium-to-high-throughput system using a robotic liquid handler dispensing into 96-well microtitre plates. This uses an automated microscope to detect DNA-stained parasite kinetoplasts in primary cell cultures from skin, muscle, liver and macrophages, and quantifies the number of T. cruzi per host cell. Screening capacity has risen from 100 compounds a month to 1,000 compounds a week in whole-cell assays, or tens of thousands a week in enzyme biochemical assays.

Fred Buckner at the University of Washington Seattle in the United States has developed an HTS based on 384-well plates and recombinant T. cruzi expressing fluorescent β-galactosidase: “We’ve gone from an era where you could test a handful of compounds in a week to [one where it is possible to test] 250,000 in a few weeks. It’s helping everybody who’s interested in screening drugs — a much needed tool.” He has shared the system with laboratories in Brazil, Argentina, Panama, the United States and Europe.

LUCIO FREITAS-JUNIOR

Images of an osteoblast human cell line infected with Trypanosoma cruzi before (left) and after (right) software detection of cell boundaries.

One of the fastest HTS systems belongs to Lucio Freitas-Junior at the Institute Pasteur in Korea: a 384-well cell-culture system that combines an automated confocal microscope with an image analyser (pictured) to screen up to 30,000 compounds a week. Through DNA staining of wild-type rather than genetically modified T. cruzi, the system’s software can count the number of parasites per cell and the number of cells per well, measure the size and shape of each cell to give a read-out of viability and morphological change owing to parasite infection, and determine the effects of the would-be inhibitors. A second step selects compounds that interfere with replication of parasite kinetoplast DNA (which provides a specific set of targets that are relatively similar among T. cruzi, Trypanosoma brucei and Leishmania). Freitas-Junior and colleagues are now screening a Pfizer library of 150,000 compounds for leads against Leishmania and T. cruzi with a commission from the DNDi.

The HTS technology “has not been applied broadly in support of drug discovery for neglected diseases, so it presents a great opportunity to evaluate a large number of compounds to generate starting materials for further analysis”, says Solomon Nwaka of the WHO–TDR. Nonetheless, Nwaka is under no illusions about the difficulty of moving to the next state. “Just because you have a million compounds available to screen, doesn’t mean you will get a new drug. HTS just gets you to the next step”.

source: nature.com