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. 2013 Feb 27;3(2):120158.
doi: 10.1098/rsob.120158.

Yeast-based Automated High-Throughput Screens to Identify Anti-Parasitic Lead Compounds

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Free PMC article

Yeast-based Automated High-Throughput Screens to Identify Anti-Parasitic Lead Compounds

Elizabeth Bilsland et al. Open Biol. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Fluorescence labelling of yeast strains. (a) Wild-type yeast transformed with plasmids expressing Venus (yellow fluorescent protein), Sapphire (blue fluorescent protein), mCherry (red fluorescent protein) or CFP (cyan fluorescent protein); visualized under bright field (BF) or ultraviolet (UV) light. (b) Schematic view of the experimental design developed for high-throughput screens: yeast strains expressing heterologous drug-resistant Plasmodium falciparum DHFR (PfRdhfr), Schistosoma mansoni DHFR (SmDHFR), human DHFR (HsDHFR) or P. falciparum DHFR (PfDHFR) growing in the presence of candidate anti-parasitic drugs. (c) Pictures of fluorescently labelled yeast strains (expressing the indicated heterologous DHFRs) grown in competition in the presence or absence of the anti-malarial pyrimethamine.
Figure 2.
Figure 2.
Relative fluorescence measure detected using a BMG Optima plate reader at 580 nm (excitation)/612 nm (emission) (Cherry), 405 nm (excitation)/510 nm (emission) (Sapphire), 500 nm (excitation)/540 nm (emission) (Venus) and 440 nm (excitation)/490 nm (emission) (CFP) of pooled yeast strains grown for 24 h in the presence of 0 to 500 μM pyrimethamine and 0 or 5 μg ml−1 of doxycycline. This plate reader has a limited dynamic range, and a higher-resolution instrument was used for the high-throughput screens. Plasmodium falciparum DHFR (PfRdhfr) labelled with Venus, S. mansoni DHFR (SmDHFR) labelled with CFP, human DHFR (HsDHFR) labelled with mCherry and P. falciparum DHFR (PfDHFR) labelled with Venus.
Figure 3.
Figure 3.
Example of a high-throughput screening result. Relative fluorescence measure detected using a BMG Polarstar plate reader at 580 nm (excitation)/612 nm (emission) (Cherry), 405 nm (excitation)/ 510 nm (emission) (Sapphire) and 500 nm (excitation)/540 nm (emission) (Venus) of pooled yeast strains grown in the presence of 5 μg ml−1 of doxycycline AND 10 μM pyrimethamine, 10 μM of test compound or no drug. Plasmodium vivax DHFR labelled with Sapphire (blue squares), human DHFR labelled with mCherry (red squares) and drug-resistant P. falciparum DHFR (PfRdhfr) labelled with Venus (yellow squares).
Figure 4.
Figure 4.
Network of connections between kinetoplastid targets and our hits. Overview of the compounds from the Maybridge hitfinder library identified as specific inhibitors of the following parasite (Trypanosoma brucei, Tb; T. cruzi, Tc; Leishmania major, Lm) targets: dihydrofolate reductase (DHFR), blue; N-myristoyltransferase (NMT), red; phosphoglycerate kinase (PGK), green. Small nodes represent hits; yellow nodes represent compounds tested in T. brucei cultures. Diamond nodes represent compounds active in Trypanosoma at 10 µM. Large diamonds represent compounds active in T. brucei cultures at 1 µM or less. The thickness of the edges (lines connecting the targets and compounds) represents the strength of the inhibition (thicker lines indicate stronger inhibition of growth of the yeast expressing the indicated parasite target by the connecting compound).
Figure 5.
Figure 5.
Antiplasmodial hits. (a) Network of connections between drug targets and our Plasmodium hits. Overview of the compounds from the Maybridge hitfinder library identified as specific inhibitors of the following parasite (P. falciparum, Pf, square nodes; P. vivax, Pv, large circular nodes) targets: dihydrofolate reductase (DHFR), blue; drug-resistant dihydrofolate reductase (Rdhfr), light blue; N-myristoyltransferase (NMT), yellow; phosphoglycerate kinase (PGK), green. Small nodes represents hits with Tanimoto distance coefficients of  less than 0.4 (red), less than 0.5 (pink) or greater than 0.5 (blue) to Chemblntd compounds identified in P. falciparum in vitro screens (www.ebi.ac.uk/chemblntd). (b) Chemblntd compounds (compounds with demonstrated activity against P. falciparum in ex vivo assays) and similar hits identified in our screens as potentially targeting PvPGK. Schematic depicting the structures of PvPGK-specific compounds identified in screens and similar compounds with demonstrated activity against P. falciparum.

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