Table 1.

Comparison of Assay Methods Used to Assess GLS Inhibitors

compds tested	glutamine conc	phosphate conc	enzyme	preincubation time	detection method	IC₅₀ (μM) for BPTES	additional in vitro assays conducted	ref(s)
RPTES, 4-7	7 mM	160 mM	hKGA₁_₆₆₉	20 min	GDH; formazan formation measured by absorbance at 570 nm	0.19; 0.58		28
8-10_; 28; 29	2 mM	45 mM	hKGA_124-669	0 min	[³H]-glutamate	3.3	P493 proliferation	22; 41
CB-839, 11-25; 30; 31	10 mM	150 mM	GAC	0 or 60 min	GDH; NADH formation measured by absorbance at 340 nm	0.1 (60 min) 0.2 (0 min)	P493/MDA-MB-231 /HCC1806 proliferation	44
26; 27; 52-67	13 mM or 1.8 mM	100 mM	GAC	60 min	GDH-diaphorase; resorufin formation measured at ex544/em590 nm	0.1-0.5		45; 50-52
32-36; 68; 101-104	10 mM	unknown	mouse brain/kidney homogenate	0 min	ammonia formation detected by Nessler s reagent by absorbance at 450 nm	not tested	MDA-MB-231 proliferation	46; 56
69-72	no biochemical GLS assay data reported						BT20 cellular glutaminase	53
37-51; 105-106	1 mM	50 mM	GAC	10 min	GLOD-horseradish peroxidase; resorufin formation measured at ex530/em590 nm	not tested	A549 proliferation	47—49; 57
73-87	50 mM	100 mM	hKGA₆₃_₆₆₉	15 min	GLOD-horseradish peroxidase; resorufin formation measured at ex535/em590 nm	not tested	PC3 cellular glutaminase; NCI-H1703 proliferation	23-27
88-100	18 mM	135 mM	GAC	unknown	GDH; NADH formation measured by absorbance at 340 nm	0.4	MDA-MB-231 proliferation	54