High-Throughput Screening Reveals New Glutaminase Inhibitor Molecules

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.