Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds

Abstract

We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains’ expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our ‘hits’ have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness.

Yeast: bridging the gap between phenotypic and biochemical assays for high-throughput screening

“…However, the first report of the use of a substitution platform in HTS was against the ornithine decarboxylase (ODC), a key regulatory enzyme in eukaryotic polyamine (PA) biosynthesis, from Haemonchus contortus, a nematode worm that is a major parasite of ruminants. Similar to above, the platform utilized a S. cerevisae ODC deficient mutant complemented by expression of the H. contortus orthologue [35] [36,37,38]. An S. cerevisiae substitution platform has also been used in HTS to identify inhibitors of the anti-leishmanial drug target inositol phosphorylceramide (IPC) synthase [39,40,41].…”

“…However, a fluorescent readout provides considerably greater sensitivity [50] and has been recently employed in yeast-based HTS. For example, Bilsland et al (2013) employed fluorescent tags to facilitate multiplexing, thus allowing compounds to be screened against protozoan DHFR, NMT or PGK (Table 2) simultaneously with the human orthologue in a single well of a 384-well plate [37]. More recently, fluorescein di-β-D-glucopyranoside (FDGlu; a highly sensitive substrate for the glucosidases found in the yeast cell wall) was utilized to measure yeast growth in a HTS campaign against the Leishmania IPC synthase.…”

“…In assays designed to identify anti-infectives, secondary assays often involve establishing activity against the pathogen. For example, of the specific hits identified by Bilsland et al (2013) in the multiplexed fluorescent S. cerevisiae substitution platform, 50% proved cytotoxic to the protozoan parasite Trypanosoma brucei in cellulo [37]. However, this did not directly prove that the target was attenuated, in other work approaches involving in vitro and cell-based assays have demonstrated that the anti-parasitic hits generated from yeast-based screens do actually hit the intended target [21,38,41].…”

Yeast-based systems for tropical disease drug discovery

“…Under these circumstances, 36 specific hits were identified and re-tested against cell cultures of the bloodstream parasite T. brucei (the etiological agent of sleeping sickness in Africa), with 18 of them being confirmed to be also lethal for this pathogen. [20] This approach has been developed to test eight different Brugia malayi (a parasitic worm responsible of lymphatic filariasis) drug targets. Nine out of 400 compounds effectively inhibited some of the targets, and from those 9 compounds, 5 had antifilarial activity in vitro.…”

The investigation of the “chemical structure – antihypoxic action” dependence in a series of indole and 2-oxindole derivatives containing the ethylamine fragment

“…Проте вивчення таких комплексних властивостей пов'язане з низкою проблем, вирішення яких залежить від інтенсивних теоретичних та експериментальних робіт в області біохімії, молекулярної біології, геноміки, комп'ютерного моделювання, органічного синтезу, медицини та фармакології [2]. Найбільше практичне значення такі дослідження мають для розробки нових лікарських субстанцій, які переважно є синтетичними молекулами [3].…”