Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi

Abstract

Background: Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7-15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.

Methodology/principal findings: We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.

Conclusions/significance: We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident that we can extend our efforts to the construction of strains with further filarial targets (in particular for those species that cannot be cultivated in the laboratory), and perform high-throughput drug screens to identify specific inhibitors of the parasite enzymes. By establishing synergistic collaborations with researchers working directly on different parasitic worms, we aim to aid antihelmintic drug development for both human and veterinary infections.

Fig 1. CLUSTAL 2.1 multiple sequence alignment of diphospho mevalonate decarboxylases from Brugia malayi (BmMVD), Loa loa (LlMVD), Homo sapiens (HsMVD) and Saccharomyces cerevisiae (ScMVD), highlighting two regions in the published BmMVD sequences that diverge from the consensus between the orthologous protein sequences.

These could be due to the presence of different splice variants of the Brugia enzyme, natural insertions or errors in the published sequence.* conserved residues;: chemically conserved changes;. non-conserved changes.

Fig 2. Partial CLUSTAL 2.1 multiple sequence alignment of publicly available Brugia malayi (Bm1_48165), Homo sapiens (HsSAH), Saccharomyces cerevisiae (ScSAH), and our cloned Brugia malayi (BmSAH) S-adenosyl homocysteinases, showing the “insert” missing from the publicly available nematode protein sequence.

The absence of the 27 amino acids in the publicly available Brugia sequence (Bm1_48165) could indicate a splice variant of the enzyme or simply a problem with the genome assembly.

Fig 3. Functional complementation of yeast deletions by Brugia malayi (Bm) or Homo sapiens (Hs) orthologous genes.

Relative growth of yeast strains expressing the parasite or human target compared to the growth of yeast strains expressing the native gene. Blue bars indicate the full expression of the heterologous genes from the TetO2 promoter, and red and green bars indicate the growth of strains with a reduced expression of the essential enzyme.

Fig 4. Scatter plot showing no correlation between the percentage identity between the human (Homo sapiens) or Brugia malayi proteins that do or do not complement the essential functions of the yeast (Saccharomyces cerevisiae) orthologues.

The average protein identity (in %) between heterologous proteins able to complement the yeast deletions was 52.5 ± 10.6, whereas the identity between heterologous proteins NOT able to complement the yeast deletions was 48.1 ± 10.0.

Fig 5. Validation of the hit compounds using Brugia pahangi adult female worms.

Chemical structures were obtained from www.chemspider.com. The IC₅₀ for each of the compounds in human fibroblasts (MRC5) is shown in the last column [26]. The IC₅₀ of Geldanamycin against different human cell lines is a consensus of multiple data available in the literature (www.medchemexpress.com/Geldanamycin.html).