Glutaminase And The Anticancer Activity Of Its Inhibitors

Mutations can lead to a significant alteration in the glucose metabolism pathway.For example,the muscle isoenzyme splice variant of pyruvate kinase can cause the Warburg effect in the glycolysis;cancer cells utilize anaerobic pathway to burn most glucose in a dissipative way to produce ATP to maintain their own metabolic needs regardless of oxygen availability;Simultaneously,the glutaminolosis is upregulated in the mitochondria,glutamine is is converted to glutamate by glutaminase,then to ?-ketoglutarate by glutamate dehydrogenase,and finallyentering the TCA cycle for ATP and NAD?P?H synthesis.Glutaminase is an emerging and important drug target for cancer.which controls glutaminolysis and also tumor cell growth.In this paper,the human kidney-type glutaminase was successfully cloned and expressed in E.coli.We also developed its activity analysis based on the new EZMTT reagent.Furthermore,100 glutaminase inhibitors were screened,and ebselen was identified as a GDH inhibitor but not a glutaminase inhibitor,and HJ7 was identified as a dual specific KGA/GDH inhibitor.The details of studies are listed below:1.Human kidney-type KGA gene was PCR amplified from A549 c DNA.The purified PCR product was restriction digested and ligated into the pET 22 b vector to generate the pET-hKGA plasmid with C-terminal his-taq.After DNA sequencing,the plasmid was transformed into an E.coli BL 21 strain.The cells were inoculated in 1:20 ratio.when OD600 reached 0.4 the expression of the hKGA protein was induced by adding 1 mM IPTG for 3 hrs.The resulting protein was purified using nickel affinity chromatographyand analyzed by SDS-PAGE which showed a single band with a molecular weight of 56 kDa.2.Based on the tetrazolium-formazan-NAD?P?H system,a novel mono-sulfonated tetrazolium salt?EZMTT?was synthesized and used to develop glutamate dehydrogenase assay.EZMTT showed essentially no absorbance at 450 nm,but the corresponding reduced form?formazan?had strong absorbance at 450 nm;EZMTT has less oxidative activity than WST-8,which makes the EZMTT reagent more compatible with commonly used antioxidants such as BME or EGCG in assay buffers,and generates less detection false positives.EZMTT is compatible withup to 0.2% Tween 20 or 1% SDS.In comparison with WST-8,EZMTT detection reagent was more stable.For application of the EZMTT detection system,E.coli GDH with N-terminal his-taq was cloned and the GDH activity assay was developed using EZMTT and further the KGA/GDH coupled assay.3.Compared the traditional glutaminase inhibition assay?GO coupled assay?with the KGA/E.coli GDH coupled assay using EZMTT as detection reagent.The GO coupled assay showed that Ebselen was a potent KGA inhibitor.However,we used paper chromatography and discovered that Ebselen did not show inhibition of KGA.Further GDH assay showed Ebselen is a GDH inhibitor.For more direct and stringent characterization of specific glutaminase inhibitors,we test the inhibition of glutamate dehydrogenase,GO,HRP,H₂O₂ one by one.HPLC showed Ebselen can react with H₂O₂;the HRP inhibition assay showed that Ebselen inhibited HRP.Therefore,Ebselen as an glutaminase inhibitor is a false positive caused by the GO coupled assay.4.The KGA/E.coli GDH coupled assay with EZMTT was applied to screening for potent KGA inhibitors.100 glutaminase inhibitors were synthesized in our lab.Among them,the inhibition by compound HJ 2 was slightly weaker than BPTES;compound HJ 7 could inhibit both KGA?IC50 was 0.2 ?M,0.08 ?M,respectively?and E.coli GDH?IC50 was 0.32 ?M,0.035 ?M,respectively?;HJ 14 had good inhibition with GDH?IC50 0.035 ?M?.What's more,compound HJ 3,HJ 8,HJ 9,HJ 15,HJ 48,HJ 49,HJ 50,HJ 51,HJ 60,HJ 81 all showed some inhibition in glutaminase and glutamate dehydrogenase coupled assays.