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.
Highlights d Genetics and chemo-proteomics identify the target of a promising anti-leishmanial d Biochemical assays confirm the direct inhibition of oxidosqualene cyclase in cells d Docking and modeling studies identify key interactions between compound and target d Strategies to improve the potency of this benzothiophene are proposed
The
glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate,
an important anaplerotic source for the tricarboxylic acid cycle in
rapidly growing cancer cells under the Warburg effect. Glutamine-derived
α-ketoglutarate is also an important cofactor of chromatin-modifying
enzymes, and through epigenetic changes, it keeps cancer cells in
an undifferentiated state. Moreover, glutamate is an important neurotransmitter,
and deregulated glutaminase activity in the nervous system underlies
several neurological disorders. Given the proven importance of glutaminase
for critical diseases, we describe the development of a new coupled
enzyme-based fluorescent glutaminase activity assay formatted for
384-well plates for high-throughput screening (HTS) of glutaminase
inhibitors. We applied the new methodology to screen a ∼30,000-compound
library to search for GLS inhibitors. The HTS assay identified 11
glutaminase inhibitors as hits that were characterized by in silico, biochemical, and glutaminase-based cellular assays.
A structure–activity relationship study on the most promising
hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In
summary, we discovered a new glutaminase inhibitor with an innovative
structural scaffold and described the molecular determinants of its
activity.
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