| In Vitro | In vitro activity: BPTES inhibits glutaminase activity expressed in human kidney cells with IC50 of 0.18 μM, and inhibits glutamate efflux by microglia with IC50 of 80-120 nM. BPTES preferentially slows cell growth in D54 cells with mutant IDH1. BPTES also inhibits glutaminase activity, lowers glutamate and α-KG levels, and increases glycolytic intermediates. BPTES (10 μM) inhibits cell growth of mHCC 3–4 cells derived from LAP/MYC tumors. BPTES also inhibits growth of a MYC-dependent P493 cells by blocking DNA replication, leading to cell death and fragmentation.
Kinase Assay: Assay plates are prepared containing 2 μL test compound in DMSO/well. The enzyme is diluted to 1 unit (liver) or 0.8 unit (kidney)/100 μL in glutaminase assay buffer, and 100 μL diluted enzyme is added to each well of the assay plate by Multidrop. The contents are mixed by shaking at full speed for 1 min on TiterMix 100. The plates are preincubated at room temperature (RT) for 20 min to allow binding of test compounds to glutaminase, and 50 μL glutamine solution (7 mM in assay buffer) is added to each well by Multidrop. The contents are shaken at full speed for 30 sec on TiterMix 100, and the plates are then incubated at RT for 60 min (liver) or 90 min (kidney). To stop the reactions, 20 μL HCl (0.3 N) is added to each well by Multidrop and mixed immediately by shaking for 30 sec on TiterMix 100. For quantification, glutamate (formed by glutaminase-catalyzed hydrolysis of glutamine) is oxidized to 2-oxoglutarate by a second enzyme, glutamate dehydrogenase (GDH), with the concomitant production of the reduced form of nicotinamide adenine dinucleotide (NADH). Reduction of nitro blue tetrazolium (NBT) in the assay solution by NADH, catalyzed by phenazine methosulphate (PMS), results in the formation of a blue-purple formazan. The absorption of formazan at 540 nm is linearly proportional to the concentration of glutamate up to 200 μM. NBT/GDH reagent (50 μL) is added to each well by Multidrop and mixed by shaking for 30 sec on TiterMix 100, and the plates are incubated at RT for 20 min to allow color formation by the GDH reaction. Glutamate concentration is determined from formazan concentration as determined by reading OD540 nm on a SpectraMax 340.
Cell Assay: Cell lines with mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were used. In all IDH1-mutant AML cells, compared with DMSO, exposure to 20 μmol/L BPTES reduced the cell growth by approximately 50% on day 4. 20 μmol/L BPTES was not significantly different from 40 μmol/L BPTES in the reduction effect. Treatment without drug was not significantly different from treatment with DMSO in the growth of cells. BPTES did not significantly affect the cell growth of wild type AML cells [3]. In tumor cells, BPTES inhibited the conversion of glutamine into glutamate. |
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