Rapid Chagas Disease Drug Target Discovery Using Directed Evolution in Drug-Sensitive Yeast

Abstract

Recent advances in cell-based, high-throughput phenotypic screening have identified new chemical compounds that are active against eukaryotic pathogens. A challenge to their future development lies in identifying these compounds’ molecular targets and binding modes. In particular, subsequent structure-based chemical optimization and target-based screening require a detailed understanding of the binding event. Here we use directed evolution and whole-genome sequencing of a drug-sensitive S. cerevisiae strain to identify the yeast ortholog of TcCyp51, lanosterol-14-alpha-demethylase (TcCyp51), as the target of MMV001239, a benzamide compound with activity against Trypanosoma cruzi, the etiological agent of Chagas disease. We show that parasites treated with MMV0001239 phenocopy parasites treated with another TcCyp51 inhibitor, posaconazole, accumulating both lanosterol and eburicol. Direct drug-protein binding of MMV0001239 was confirmed through spectrophotometric binding assays and X-ray crystallography, revealing a binding site shared with other anti-trypanosomal compounds that target Cyp51. These studies provide a new probe chemotype for TcCyp51 inhibition.

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway

“…For target identification, we first used in vitro evolution studies in the model system Saccharomyces cerevisiae, a strategy that has been used to discover the target of cladosporin, a tRNA synthetase inhibitor that acts against both P. falciparum and S. cerevisiae. We have recently demonstrated that yeast can be successfully used for drug target identification in both antimalarial and antitrypanosomal compounds [28][29][30] , so we decided to perform selections with GNF179. IZPs are moderately active (45 μM) against an attenuated strain of Saccharomyces that has been genetically modified by replacing 16 ABC multi-drug transporter genes with modified Aequorea victoria GFP (eGFP) and that has been dubbed the "Green Monster" 31 .…”

Glutamine metabolism modulates azole susceptibility inTrypanosoma cruziamastigotes

“…This species, or collection of species, co-migrated with the ergosterol standard. However, T. cruzi amastigotes reportedly do not generate canonical ergosterol as the final species from endogenous sterol synthesis (Gunatilleke et al, 2012; Liendo et al, 1999; Ottilie et al, 2017).…”

“…As expected, carbons from exogenous 13 C-glutamine were readily incorporated into species downstream of CYP51 (Figure 3B). In the presence of ketoconazole, when CYP51 is inhibited, these downstream species were absent, and 13 C was incorporated into lanosterol and ebericol (Figure 3B,C), the two species immediately upstream of CYP51 in the sterol synthesis pathway (Gunatilleke et al, 2012; Ottilie et al, 2017). Unlike endogenous sterols and synthesis intermediates derived from amastigote metabolism, we did not identify any incorporation of 13 C-glutamine into host derived, but amastigote-associated, cholesterol (Figure 3B,C).…”

Pan-active imidazolopiperazine antimalarials target thePlasmodium falciparumintracellular secretory pathway

“…CRISPR- Cas 9 genome engineering was performed on the S. cerevisiae ABC 16 -Monster strain using vectors p414 and p426 obtained from the Church lab (Addgene) as previously described 72 . To produce gRNA plasmids specific to the desired mutation sites, oligonucleotides were synthesized (Integrated DNA Technologies) to match the target sequence and contain a 24 base-pair overlap with the p426 vector backbone.…”