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Comparison of the effects of UPGL00004, BPTES, and CB-839 on the growth of breast cancer cells. A, BPTES, CB-839, and UPGL00004 were added to MDA-MB-231 cells in the indicated amounts and their effects on cell proliferation were assayed. B, HS578T, MDA-MB-231, TSE, and MDA-MB-453 cells were treated in serum-free media for 14 h with BPTES, CB-839, or UPGL00004 (UPGL4). The media was then collected, and the amount of ammonia generated was determined for each sample. Error bars for panels A and B represent the mean S.D. of three independent experiments. C, plots showing mean tumor volumes (mm 3 ) as a function of time for mice containing triple-negative HCI-002 grafts, which were treated with the different drug combinations. When tumors reached 3 mm in diameter, IP injections with vehicle alone (control) (black diamonds), bevacizumab (2.5 mg/kg weight of the animal) (white triangles), UPGL00004 (1 mg/kg weight of the animal) (white circles), or the two drugs together (gray squares), were injected every other day for 4 weeks. The tumor volumes for the bevacizumab plus UPGL00004 (n 6), versus treatment with bevacizumab (n 6), UPGL00004 (n 6), or DMSO alone, were statistically significant. Error bars represent the S.E. for this experiment.
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Altered glycolytic flux in cancer cells (the Warburg effect) causes their proliferation to rely upon elevated glutamine metabolism (glutamine addiction). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small mole...
Citations
... It provides material and energy bases for tumor growth, proliferation, invasion, and metastasis. Studies suggest that AAT usually induces hypoxia microenvironment before tumoracquired drug resistance, which is beneficial to the transformation of tumor metabolism to glycolysis and glutamine metabolism (48)(49)(50). In VC, the tumor tissue showed enhanced glycolysis and pentose phosphate pathway activation (51). ...
Tumor vasculature is pivotal in regulating tumor perfusion, immune cell infiltration, metastasis, and invasion. The vascular status of the tumor is intricately linked to its immune landscape and response to immunotherapy. Vessel co-option means that tumor tissue adeptly exploits pre-existing blood vessels in the para-carcinoma region to foster its growth rather than inducing angiogenesis. It emerges as a significant mechanism contributing to anti-angiogenic therapy resistance. Different from angiogenic tumors, vessel co-option presents a distinctive vascular-immune niche characterized by varying states and distribution of immune cells, including T-cells, tumor-associated macrophages, neutrophils, and hepatic stellate cells. This unique composition contributes to an immunosuppressive tumor microenvironment that is crucial in modulating the response to cancer immunotherapy. In this review, we systematically reviewed the evidence and molecular mechanisms of vessel co-option in liver cancer, while also exploring its implications for anti-angiogenic drug resistance and the immune microenvironment, to provide new ideas and clues for screening patients with liver cancer who are effective in immunotherapy.
... In GAC, this loop contains the binding site for the BPTES-class of allosteric inhibitors which includes CB-839, a drug candidate that has been in clinical trials 26 . The loop forms a stable, inactive conformation when bound to BPTES and to its more potent analogs, CB-839 (not shown) and UPGL0004 35,36 (Supplementary Fig. 1). However, the activation loop of GLS2 differs from KGA and GAC by two residues, rendering GLS2 insensitive to these inhibitors 24 . ...
... An N-terminal His-tagged form of the full-length human liver-type glutaminase isoform GLS2 without the mitochondrial localization sequence (residue 38-602) was cloned into the pET28a plasmid 36 . Sitedirected mutagenesis was performed using Phusion DNA polymerase (New England Biolabs, NEB). ...
The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the ‘glutamine addiction’ of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an ‘activation loop’ to assume a specific conformation that works together with a ‘lid’ to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.
... The activation loop (L321 m -RFNKL-F327 m ; Fig. 1d and Extended Data Fig. 2a,b) is generally unresolved in crystal structures, except when bound to an allosteric inhibitor 9,13 . At the monomer level, although the backbone tracing of fGLS m was virtually identical to, for instance, those of ligand-free mouse GAC (PDB 3SS3) and human GAC bound to an allosteric inhibitor (PDB 5HL1 (ref. ...
... Allosteric inhibitors, such as CB-839 (ref. 13) or BPTES 10 , stabilize the tetramer in a rigid inactive state through the expansion of dimer-and tetramer-forming interfaces 10,13 . c, Electrostatic potential surfaces highlight the unique presence of a very large electropositive region at the tetramer interface of fGLS m (yellow arrows) that favors the binding of the negatively charged phosphate ions. ...
... Allosteric inhibitors, such as CB-839 (ref. 13) or BPTES 10 , stabilize the tetramer in a rigid inactive state through the expansion of dimer-and tetramer-forming interfaces 10,13 . c, Electrostatic potential surfaces highlight the unique presence of a very large electropositive region at the tetramer interface of fGLS m (yellow arrows) that favors the binding of the negatively charged phosphate ions. ...
Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.
... In a recent study, Udupa et al. [71] showed that treatment of patient-derived pancreatic orthotopic JHU094 tumors in male Foxn1 nu athymic nude mice with BPTES (bis-2-(5phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide) encapsulated in nanoparticles (BPTES-NP) resulted in a large increase in endogenous KGM. (JHU is an acronym for Johns Hopkins University; BPTES is an inhibitor of allosteric binding of phosphate to GLS1/GAC [72][73][74][75][76]). The increase is presumably due, at least in part, to the increased levels of L-glutamine available for transamination. ...
... The Johns Hopkins group have encapsulated BPTES in nanoparticles and shown that, in combination with metformin, it is effective against pancreatic cancer in a mouse model [249]. The Cerione group at Cornell University has been instrumental in studying the mechanism of inhibition of GLS1/GAC by BPTES and in characterizing additional compounds related to BPTES, which may prove useful in future clinical trials [73][74][75][76]. According to Milano et al., over 2000 BPTES analogues have been synthesized [76]. ...
Many cancers utilize l-glutamine as a major energy source. Often cited in the literature as “l-glutamine addiction”, this well-characterized pathway involves hydrolysis of l-glutamine by a glutaminase to l-glutamate, followed by oxidative deamination, or transamination, to α-ketoglutarate, which enters the tricarboxylic acid cycle. However, mammalian tissues/cancers possess a rarely mentioned, alternative pathway (the glutaminase II pathway): l-glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. The name glutaminase II may be confused with the glutaminase 2 (GLS2) isozyme. Thus, we recently renamed the glutaminase II pathway the “glutamine transaminase—ω-amidase (GTωA)” pathway. Herein, we summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway is that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia, diminishing cellular energy demands. We suggest that the ability to coordinate control of both pathways bestows a metabolic advantage to cancer cells. Finally, we discuss possible benefits of GTωA pathway inhibitors, not only as aids to studying the normal biological roles of the pathway but also as possible useful anticancer agents.
... For instance, by reshaping the balance between Th17 and Treg cells, selective inhibition of GOT1 with (aminooxy)acetic acid (AOA) ameliorates experimental autoimmune encephalomyelitis in mice (30). Several GLS1 inhibitors (BPTES, compound 968, CB-839), compound 19 and UPGL00004 have also been reported to inhibit GAC (87,(89)(90)(91)(92). Besides, IDH1, the primary catalytic enzyme for 2-HG synthesis and a key regulator of naive CD4 + T cell differentiation, has enormous promise as a target for therapeutic intervention. ...
To maintain the body’s regular immune system, CD4⁺ T cell homeostasis is crucial, particularly T helper (Th1, Th17) cells and T regulatory (Treg) cells. Abnormally differentiated peripheral CD4⁺ T cells are responsible for the occurrence and development of numerous diseases, including autoimmune diseases, transplantation rejection, and irritability. Searching for an effective interventional approach to control this abnormal differentiation is therefore especially important. As immunometabolism progressed, the inherent metabolic factors underlying the immune cell differentiation have gradually come to light. Mounting number of studies have revealed that glutaminolysis plays an indelible role in the differentiation of CD4⁺ T cells. Besides, alterations in the glutaminolysis can also lead to changes in the fate of peripheral CD4⁺ T cells. All of this indicate that the glutaminolysis pathway has excellent potential for interventional regulation of CD4⁺ T cells differentiation. Here, we summarized the process by which glutaminolysis regulates the fate of CD4⁺ T cells during differentiation and further investigated how to reshape abnormal CD4⁺ T cell differentiation by targeting glutaminolysis.
... In a recent study, Udupa et al. [71] showed that treatment of patient-derived pancreatic orthotopic JHU094 tumors in male Foxn1 nu athymic nude mice with BPTES (bis-2-(5phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide) encapsulated in nanoparticles (BPTES-NP) resulted in a large increase in endogenous KGM. (JHU is an acronym for Johns Hopkins University; BPTES is an inhibitor of allosteric binding of phosphate to GLS1/GAC [72][73][74][75][76].) The increase is presumably due, at least in part, to the accumulation of increased levels of L-glutamine available for transamination. ...
... The Johns Hopkins group have encapsulated BPTES in nanoparticles and shown that, in combination with metformin, it is effective against pancreatic cancer in a mouse model [249]. The Cerione group at Cornell University has been instrumental in studying the mechanism of inhibition of GLS1/GAC by BPTES and in characterizing additional compounds related to BPTES which may prove useful in future clinical trials [73][74][75][76]. According to Milano et al., over 2,000 BPTES analogues have been synthesized [76]. ...
Many cancers utilize L-glutamine as a major energy source. This “L-glutamine addiction” involves a well-characterized pathway whereby L-glutamine is hydrolyzed by a glutaminase (GLS) to L-glutamate, which is then converted to α-ketoglutarate, the carbons of which enter the tricarboxylic acid (TCA) cycle. However, mammalian tissues/cancers possess a rarely mentioned alternative pathway (the glutaminase II pathway): L-Glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. Uncertainty may prevail over the name glutaminase II which may be confused with the enzyme named glutaminase 2 (GLS2). Thus, we recently suggested a new name for the glutaminase II pathway, namely the glutamine transaminase-ω-amidase (GTωA) pathway. Herein, we 1) evaluate three recent articles that mention L-glutamine addiction, but not the GTωA pathway, 2) summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and 3) as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway [i.e., L-glutamine + α-keto acid + H2O α-ketoglutarate + L-amino acid + +NH4] is that it is irreversible and that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia. Finally, we discuss possible clinical benefits of GTωA pathway inhibitors.
... It is reported that in urine and serum samples from patients with COVID-19 and healthy controls, the relative abundance of 301 proteins showed opposite expression patterns in urine and serum as the disease progressed [141]. Serum proteins also vary in DLBCL patients with different prognoses, as mentioned in Section 2. [137] Metformin AZD3965 [134] BPTES [135] CB-839 [136] Fasnall [137] Metformin [138] Ritonavir [139] Temsirolimus [140] 6. Summary ...
... It is reported that in urine and serum samples from patients with COVID-19 and healthy controls, the relative abundance of 301 proteins showed opposite expression patterns in urine and serum as the disease progressed [141]. Serum proteins also vary in DLBCL patients with different prognoses, as mentioned in Section 2. [138] Ritonavir AZD3965 [134] BPTES [135] CB-839 [136] Fasnall [137] Metformin [138] Ritonavir [139] Temsirolimus [140] 6. Summary ...
... It is reported that in urine and serum samples from patients with COVID-19 and healthy controls, the relative abundance of 301 proteins showed opposite expression patterns in urine and serum as the disease progressed [141]. Serum proteins also vary in DLBCL patients with different prognoses, as mentioned in Section 2. [139] Temsirolimus AZD3965 [134] BPTES [135] CB-839 [136] Fasnall [137] Metformin [138] Ritonavir [139] Temsirolimus [140] 6. Summary ...
Cells utilize different metabolic processes to maintain their growth and differentiation. Tumor cells have made some metabolic changes to protect themselves from malnutrition. These metabolic alterations affect the tumor microenvironment and macroenvironment. Developing drugs targeting these metabolic alterations could be a good direction. In this review, we briefly introduce metabolic changes/regulations of the tumor macroenvironment and microenvironment and summarize potential drugs targeting the metabolism in diffuse large B-cell lymphoma.
... Similarly, new interactions were formed with the key residues PHE 322 (A), LEU 323 (A), and ARG 317 (B). Knowing that these amino acids were reported to have a crucial role in the allosteric site of GAC[42,44]. ...
... CB-839 54 conferred resistance to CB-839 ( Supplementary Fig. 5d-i). These results are in line with above glutamine deprivation data, supporting that glutamine dependency in SMARCA4/2-deficient cancer cells can be exploited therapeutically. ...
SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.
... This class of allosteric compound has been shown to be effective in a number of different cancers. The compounds have been most heavily studied in the context of triple-negative breast cancer, where potent compounds such as CB-839 [57] or UPGL00004 [69] can inhibit the growth of models such as MDA-MB-231 or Hs578T with nanomolar potency. However, CB-839 especially has been widely studied in other contexts, and has been demonstrated to be effective against e.g. ...
... This particular formulation of the drug relied upon solubilization with 25% w/v hydroxypropyl-cyclodextrin [57]. Another study examined injected UPGL-00004 (1 mg/kg mouse body weight), in a carrier agent consisting of RPMI-1640, 5% DMSO, 5% Cremophor EL, and 5% ethanol [69]. While this was a reduced amount of a comparable drug, it was also in a relatively aggressive injection carrier, depending upon Cremophor EL, a non-ionic surfactant which, despite its long history of pharmaceutical use to solubilize hydrophobic drugs, also has several identified negative health effects associated with it [120][121][122]. ...
Metabolic reprogramming is a major hallmark of malignant transformation in cancer, and part of the so-called Warburg effect, in which the upregulation of glutamine catabolism plays a major role. The glutaminase enzymes convert glutamine to glutamate, which initiates this pathway. Inhibition of different forms of glutaminase (KGA, GAC, or LGA) demonstrated potential as an emerging anti-cancer therapeutic strategy. The regulation of these enzymes, and the molecular basis for their inhibition, have been the focus of much recent research. This review will explore the recent progress in understanding the molecular basis for activation and inhibition of different forms of glutaminase, as well as the recent focus on combination therapies of glutaminase inhibitors with other anti-cancer drugs.
