Figure 1.

The HTS campaign was performed with a three-enzyme-based fluorescent assay. (A) Scheme of the three-enzyme-based reaction. GLS converts glutamine in glutamate, which is further converted to α-ketoglutarate by GDH. GDH generates NADH from NAD+, which is used by diaphorase to convert resazurin in resorufin, a fluorescent compound. (B) HTS statistical parameters. Above: relative fluorescent unit values of the positive (1% DMSO) and negative controls (reaction without GAC) (on the left) and the Z′ factor obtained for each plate. Below: signal-to-noise (S/N) and signal-to-background (S/B) values for each plate. (C) HTS campaign showing the calculated percentage of GLS activity on each well. The solid red line indicates average % of activity, and the dashed red line indicates 3 standard deviation (SD) of the average, the established hit limit.