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Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani , the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S) -1 -treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S) -1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)- 1 and the Ld OSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed. [Display omitted] • Genetics and chemo-proteomics identify the target of a promising anti-leishmanial • Biochemical assays confirm the direct inhibition of oxidosqualene cyclase in cells • Docking and modeling studies identify key interactions between compound and target • Strategies to improve the potency of this benzothiophene are proposed Paradela et al. investigated the mechanism of action of a promising anti-leishmanial. Using genetics, chemo-proteomics, and biochemical approaches, the target of this benzothiophene was confirmed as oxidosqualene cyclase, a key enzyme of sterol biosynthesis. Docking and modeling identified key interactions between this compound and the active site of this enzyme. [ABSTRACT FROM AUTHOR]

Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines. [ABSTRACT FROM AUTHOR]