Asparagine synthetase ASNS is a glutamine amidotransferase t

Asparagine synthetase (ASNS) is a glutamine amidotransferase that catalyzes ATP-dependent synthesis of asparagine and glutamate from aspartate and glutamine. Upregulation of ASNS expression renders leukemia cells resistant to l-asparaginase treatment, and ASNS is essential for cell survival in the absence of exogenous asparagine [31]. Overexpression of ASNS occurs in a number of solid tumors including gliomas and neuroblastomas, where it is associated with poor prognosis [31]. Combining l-asparaginase treatment with knockdown of ASNS expression simultaneously deprives cells of external and internal asparagine supplies, and potently inhibits tumor growth in vivo[32]. Attempts in the 1970s to target ASNS as a strategy for overcoming l-asparaginase resistance in leukemia cells failed to identify potent inhibitors. However, the available preclinical data indicate that further efforts to inhibit ASNS pharmacologically are justified. A product of the glutaminases and the amidotransferases is glutamate, which is present at high intracellular concentration (∼25mM in HeLa cells [33]) and plays a central part in cellular amino SB 415286 metabolism. Major fates of glutamate include: (i) incorporation into polypeptide chains during protein synthesis; (ii) ligation with cysteine to form γ-glutamylcysteine, which is subsequently condensed with glycine to yield the antioxidant glutathione; (iii) efflux through antiporters coupled to uptake of extracellular amino acids; (iv) conversion to α-ketoglutarate (α-KG), with nitrogen either released as ammonium (deamination) or transferred to an α-ketoacid to generate the corresponding amino acid (transamination); and (v) diversion into the proline synthesis pathway (Fig. 2). Of the glutamate antiporters, SLC7A11 (xCT) has received particular attention as a possible target for cancer therapy [34,35]. Glutamate efflux through SLC7A11 drives acquisition of cystine from the extracellular environment. Cystine is rapidly reduced in the cell to cysteine, which can then be used in protein and glutathione synthesis. Although the small molecules sulfasalazine, erastin and sorafenib block SLC7A11 function, each has other known targets and there is a need to develop more-selective inhibitors of this transporter [34,35]. Oxidative deamination of glutamate to α-KG is catalyzed by mitochondrial glutamate dehydrogenase 1 and 2 (GLUD1/2), and transaminations are catalyzed by several enzymes, some of which have mitochondrial and cytosolic isoforms (Fig. 2). Proliferating mammary epithelial cells catabolize glutamate primarily through transaminases, coupling α-KG generation to nonessential amino acid synthesis [36]. As cells transition to quiescence, transaminase expression decreases and GLUD expression is upregulated. Consistent with these findings, highly proliferative human breast tumors display high transaminase and low GLUD expression, with expression of the transaminase genes PSAT1 (cytosolic serine synthesis), GPT2 (mitochondrial alanine synthesis) and GOT1 (cytosolic aspartate synthesis) correlating strongly with proliferation rate [36]. Oncogenic KRAS suppresses GLUD and upregulates GOT1 expression in pancreatic ductal adenocarcinoma as part of a metabolic program that supports redox homeostasis [12], and GPT2 is overexpressed in colon tumors, driving glutamine utilization for TCA cycle anaplerosis [37,38]. However, GLUD has important roles in certain tumors, and knockdown of GLUD1 expression in lung cancer cells inhibits tumorigenesis in a xenograft model [39]. Efforts to target transaminases and GLUD are currently preclinical, and there is a notable absence of inhibitors that are selective for individual transaminases. Aminooxyacetate, a pan-transaminase inhibitor, retards tumor growth in breast cancer xenograft models [40], and the GLUD1 inhibitor R162 suppresses growth of lung cancer xenografts [39]. The branched-chain amino acid transaminase 1 (BCAT1) is important for proliferation of IDH1-wild-type glioma cells and also for non-small-cell lung carcinoma allografts [41,42]. Although the FDA-approved drug gabapentin inhibits BCAT1, its Ki is in the millimolar range and more-potent inhibitors are probably necessary for effective BCAT1 inhibition in vivo.