High-throughput screening reveals new glutaminase inhibitor molecules

[Image: see text] 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.