Glutaminase inhibition as potential cancer therapeutics: current status and future applications

Abstract

Alterations in normal metabolic processes are defining features of cancer. Glutamine, an abundant amino acid in the human blood, plays a critical role in regulating several biosynthetic and bioenergetic pathways that support tumour growth. Glutaminolysis is a metabolic pathway that converts glutamine into various metabolites involved in the tricarboxylic acid (TCA) cycle and generates antioxidants that are vital for tumour cell survival. As glutaminase catalyses the initial step of this metabolic pathway, it is of great significance in cancer metabolism and tumour progression. Inhibition of glutaminase and targeting of glutaminolysis have emerged as promising strategies for cancer therapy. This review explores the role of glutaminases in cancer metabolism and discusses various glutaminase inhibitors developed as potential therapies for tumour regression.

Keywords:

• glutaminase
• GLS
• cancer metabolism
• anticancer
• KEAP1 mutation

Introduction

Cancer is caused by several alterations in molecular and cellular pathways. Tumour cells undergo neoplastic transformation because of their high energy and building block requirements, which are primarily fulfilled by the oxygen and nutrients available in the tumour microenvironment. Warburg proposed that cancer cells reprogram cellular energy metabolism by modifying aerobic glycolysis to provide increased energy to tumour cells and that aerobic glycolysis is upregulated 200-fold in cancer cells compared with normal cells.¹ The Warburg effect results in the conversion of a significant amount of glucose to lactate, which is not utilised in the mitochondrial tricarboxylic acid (TCA) cycle but is used for ATP production.² Recently, cancer metabolism hallmarks, including the deregulation of glucose and amino acid uptake and the utilisation of alternative nutrient acquisition pathways, were further reviewed.³ Tumour cells upregulate certain amino acid intake, resulting in alternative pathway activation.

Glutamine, an abundant amino acid in blood and muscle, is a crucial source of nitrogen and carbon for lipid, protein, and nucleotide biosynthesis.⁴ Oncogenes alter the cellular and metabolic transformations during tumorigenesis, highlighting the importance of glutamine in cancer cells. The Warburg effect implies a reduction in the TCA cycle, indicating that tumour cells utilise glutamine as an alternative to carbon and nitrogen sources for cellular biosynthesis.⁵ Additionally, glutamine deficiency, rather than glucose deficiency, induces Myc-dependent apoptosis in human cells, corroborating the importance of glutamine in tumorigenesis.⁶ The essential role of glutamine in cancer cell biosynthesis and bioenergetics underscores the need for further studies on glutamine metabolism to improve cancer prognosis and develop agents that target glutamine-related pathways. This review summarises glutamine metabolism in cancer via glutaminases and their structures and provides an overview of glutaminase inhibitors and their potential to induce tumour regression.

Metabolism of glutamine in cancer

In 1990, Lacey et al. challenged the prevailing belief that glutamine is a nonessential amino acid and demonstrated its importance under catabolic stress.⁷ In tumour cells, the Warburg effect impedes the entry of pyruvate into the TCA cycle. Glutaminolysis involves the use of glutamine as a precursor to activate the TCA cycle by generating α-ketoglutarate intermediates.⁸

Glutamine plays a vital role as a cellular intermediary metabolite and can act as a carbon or nitrogen donor (α and γ-nitrogen). De novo synthesis of nucleotides requires carbon and nitrogen donor intermediates. With an increased demand for nucleotide synthesis in proliferating cells, glutamine is the preferred carbon source for pyrimidine ring biosynthesis under the hypoxic conditions of tumour cells;⁹ glutamine can also serve as a nitrogen source in the de novo synthesis of both purine and pyrimidine nucleotides in proliferating cells.¹⁰

Reductively metabolised glutamine acts as a major source of carbon for fatty acid synthesis during impaired mitochondrial respiration and hypoxia. Reductive glutamine metabolism is initiated by an increasing α-ketoglutarate/citrate ratio.¹¹ Glutamine also plays a role in acid-base homeostasis in the kidney, redox homeostasis, and reductive oxygen species (ROS) mitigation.^12,13

Glutamine plays a critical role in the growth and proliferation of rapidly dividing cells such as immune, gastrointestinal tract, and tumour cells. Glutamine deficiency can cause intestinal mucosal necrosis and apoptosis in human cell lines,^6,7 and oral administration of glutamine can improve the quality of life and promote mucosal healing in cancer patients undergoing chemotherapy and radiotherapy.¹⁴ Transporters SLC1A5 (ASCT2) and SLC7A5/SLC3A2 mediate glutamine movement across the cell membrane, and the mitochondrial variant of SLC1A5 facilitates glutamine transport across mitochondrial membranes.^15,16

Glutaminolysis involves glutamine metabolism in the mitochondria. The first step involves the conversion of glutamine to glutamate by glutaminase (Figure 1A). Glutamate then enters the TCA cycle via glutamate dehydrogenase (GLUD) or contributes to the biosynthesis of nonessential amino acids (NEAAs), such as aspartate, phosphoserine, and alanine, via aminotransferases.⁸ Glutaminolysis is crucial for maintaining redox homeostasis as it provides glutamate for glutathione (GSH) synthesis.¹⁷ The Arf1 GTPase enzyme activates the mTORC1 pathway in the lysosome through glutamine in cancer cells; hence, cancer cells show high glutamine dependence.¹⁸ A previous study reviewed the role of glutaminase isoenzymes in cancer metabolic therapy and highlighted various metabolic vulnerabilities in different types of cancers, including the correlation between glutamine metabolism and tumour malignancy.¹⁹

Figure 1. Function and structure of human glutaminases. (A) Catalytic function of glutaminase enzyme. (B) Two splice variants of GLS: KGA, the longer isoform with 18 exons, and GAC, the shorter isoform with 15 exons. (C) Two splice variants of GLS2: GAB, the longer isoform with 18 exons and LGA, the shorter isoform with 17 exons. (D, E) Structural domains of the splice variants of GLS and GLS2.

These findings suggest that glutamine metabolism in cancer cells is essential for tumour growth and progression and paves the way for studying metabolic pathway-targeted therapies for cancer treatment. The following sections provide further discussion on glutamine metabolism by the two isoforms of glutaminase: kidney-type glutaminase (GLS) and liver-type glutaminase (GLS2). These isoforms are encoded by different but related genes. GLS is expressed in various normal tissues, whereas GLS2 is restricted to the brain, pancreas, and liver.²⁰

Glutaminase

Human glutaminase isoenzymes can be categorised into GLS and GLS2 isoforms, each with long and short splice variants. KGA, the long splice variant of GLS, has exons 1–19 with a deletion of exon 15, while the short splice variant has exons 1–15 with a deletion of exons 16–19 (Figure 1B). In contrast, the long splice variant of GLS2 is GAB with exons 1–18 while the short splice variant LGA has exons 2–18 with a deletion of exon 1 (Figure 1C).²¹ The domains present in these enzymes are signal peptides at the N-terminus, the catalytic site known as the glutaminase domain, and the C-terminus (Figure 1D and E).²² Expression of these isoforms varies across different regions of the body. The GAC splice variant of GLS is highly expressed in the lungs, placenta, pancreas, and many transformed cells, whereas KGA is expressed in the brain, kidneys, intestines, and immune cells.²³ The GLS2 isoform is mainly found in the adult liver; however,²⁴ research has shown its expression in extrahepatic tissues, such as the brain and pancreas.²⁵ At the cellular level, GLS is most likely located in the mitochondria; however, the exact intramitochondrial localisation remains unclear.²⁶ Conversely, GLS2 is localised in the inner mitochondrial membrane.²⁷ Kidney-type glutaminase is involved in maintaining the acid-base balance during metabolic acidosis in the kidney,²⁸ whereas liver-type GLS2 contributes to the provision of nitrogen for the urea cycle in the liver.²⁹ The KGA variant of GLS is responsible for the production of glutamate and GABA for neurotransmission. In the intestinal epithelium, the KGA variant initiates the catabolism of glutamine as the major respiratory fuel.³⁰ In certain cancers, GLS2 is considered to function as a tumour suppressor,³¹ and its overexpression is associated with poor overall survival. However, further studies are needed to determine the exact role of GLS2 in different cancers. In contrast, recent studies have shown that GLS is frequently overexpressed in several types of cancers.²⁰ The GAC splice variant of GLS is highly expressed in triple-negative breast cancer, acute myeloid leukaemia, and non-small cell lung cancer.^32,33 These findings suggest that different glutaminase splice variants play diverse roles in various biological processes and may be involved in the development and progression of different types of cancer.

Regulators of glutaminase

Several factors have been examined to elucidate the regulation of GLS expression. GLS and GLS2 activation is strongly influenced by phosphate concentrations, with high concentrations activating GLS; however, its activity is significantly inhibited by its end product, glutamate. Conversely, GLS2 was active in low phosphate concentrations and was unaffected by the presence of glutamate.^34–36 Meanwhile, ammonia activates and inhibits GLS2.²⁰ These results offer novel insights into glutaminase enzyme regulation and indicate that different factors may have distinct effects on GLS and GLS2 activity. Further research is required to elucidate the precise mechanisms underlying these observations and their implications in cellular metabolism.

Glutaminase activity is influenced by oncogenes and epidermal growth factors (EGF). The association of the three kinase components of Rafa-1/Mek2/ERK stimulates KGA.³⁷ Myc, a proto-oncogene and a major regulator of cell proliferation, also plays a crucial role in increasing glutamine uptake and metabolism, including indirect effects on glutaminase activity.³⁸ A recent study summarised the role of Myc in suppressing miR-23a/b expression, leading to increased mitochondrial GLS expression and glutamine metabolism in PC3 prostate cancer cells and P-493B lymphoma cells.³⁹ Additionally, Myc directly binds to the E-box elements of the SLC7A5 and SLC43A1 transporters and enhances the uptake of essential amino acids, which stimulates mitochondrial glutaminolysis, leading to glutamine addiction as a bioenergetic substrate.^40,41 In human leukemic Jurkat cells, the NF-κB p65 subunit inhibits miR-23a expression, leading to increased GLS expression.⁴² Furthermore, the oncogenic transcription factor c-Jun regulates GLS expression.⁴³ These findings highlight the complex interplay between oncogenes and other factors involved in GLS regulation and suggest potential avenues for further research on the role of glutamine metabolism in cancer.

All mammalian cells express three RAS proteins (H, K, and N) that promote oncogenesis; particularly, KRAS mutations are frequently observed in cancer.⁴⁴ Studies have revealed that the KRAS and Myc oncogenes increase glutamine addiction and glycolysis, which divert glucose from the TCA cycle.⁴⁵ Additionally, nuclear factor erythroid 2 related factor 2 (NRF2) has been shown to play an important role in the metabolic reprogramming of glutamine in KRAS-mutated cells;^46,47 however, the mechanisms underlying glutamine-dependent tumour growth remain largely unknown. In KRAS-mutated non-small cell lung cancer (NSCLC), an increase in KEAP1 mutations activates the KEAP1/NRF2 pathway, leading to the increased expression of genes involved in glutamine metabolism. KRAS mutations result in the loss of serine/threonine kinase 11 (STK11) in NSCLC. The STK11 and KEAP1/NRF2 pathways drive tumour cells to become more sensitive to the glutaminase inhibitor CB-839 in in vivo and in vitro experiments.⁴⁷ Asparagine synthetase (ASNS) catalyses the formation of L-asparagine from L-aspartic acid, using L-glutamine as the nitrogen source.⁴⁸ Inhibition of GLS by IACS-6274 induces glutathione (GSH) depletion and altered cell cycle kinetics, owing to intracellular nucleotide pool depletion and DNA damage accumulation.⁴⁹ Additionally, it was observed that when determining transaminase activity with the IACS-6274 inhibitor, ASNS-low ovarian cell lines showed higher inhibition than ASNS-high cell lines, which showed resistance to the inhibitor.⁴⁹ Therefore, this study guided to ASNS as a biomarker for GLS inhibitor-based therapeutic regimens.

In many solid tumours, metabolic adaptation to hypoxic conditions is driven by hypoxia-inducible factors (HIF).⁵⁰ In normal mammalian cells, acetyl coenzyme A, a precursor of fatty acid biosynthesis, is produced from the TCA cycle via glucose-derived pyruvate. However, cell proliferation under hypoxic conditions depends primarily on the reductive carboxylation of glutamine for lipid biosynthesis.⁵¹ For example, hypoxia induces GLS expression. Under hypoxic conditions, GLS mRNA and protein expression increases via hypoxia-inducible factor 1 (HIF-1).⁵² Furthermore, in addition to transcriptional and post-transcriptional modifications, post-translational modifications (PTM) are key factors in GLS activation.^37,53 These findings suggest the complex regulation of glutamine metabolism in cancer and highlight the need for further research into its potential therapeutic implications.

In contrast to GLS, GLS2 is a p53 target gene in both normal and tumour cells,⁵⁴ and human GLS2 contains a p53 consensus DNA-binding element.⁵⁵ In addition to p53, TAp63, and TAp73 regulate GLS2 expression.^56,57 Taken together, these findings highlight the importance of the glutaminase-mediated metabolism of glutamine and its regulation in different cancers. Target-based therapies targeting glutamine-related metabolic pathways may represent a promising strategy for cancer treatment. Figure 2 provides an overview of regulators involved in glutaminase-mediated glutamine metabolism in cancer cells. This review provides a comprehensive understanding of the regulators of glutamine metabolism and their potential targets for cancer therapeutic interventions.

Figure 2. Cancer metabolism involving glutamine. Cancer cells transport glutamine through ASCT2. Glutamine thus entered is converted to glutamate through GLS and GLS2. The glutamate formed then moves into TCA cycle and supports the biosynthesis of nucleotides, proteins, and lipids. The regulators of glutaminase are marked in pink (Permission granted by Wang et al., 2020).

Inhibitors of glutaminase

Cancer cells rely on different metabolic pathways to maintain their unique characteristics such as unchecked cell proliferation, autonomous growth signalling, and resistance to apoptosis. Glutaminolysis plays a vital role in cancer development and is an attractive therapeutic target. The initial step in glutaminolysis is the conversion of glutamine to glutamate by glutaminase, making it a valuable target for cancer therapy. Inhibition of glutamine metabolism leads to a deficiency in glutathione (GSH) levels and the accumulation of mitochondrial ROS (mitoROS), ultimately leading to apoptotic cell death.^58–60 Additionally, combination therapy with glutaminase inhibitors and other chemotherapeutic agents has been proven to be effective in inhibiting tumour cell growth in ovarian and pancreatic cancers.^46,60,61 These findings highlight the potential of targeting glutaminases in cancer therapy. Several glutaminase inhibitors have been identified and classified based on their binding sites. Inhibitors that target active or catalytic sites are called orthosteric/competitive inhibitors, whereas those that target other sites are called allosteric inhibitors.

The immunosuppressive environment of tumours must be altered to achieve effective antitumor effects. The blockade of glutamine metabolism by an antagonist results in a decrease in hypoxia, acidosis, and nutrient depletion. However, this antagonism results in the upregulation of the oxidative metabolism of effector T cells, which leads to the formation of a highly activated phenotype of T effector cells.⁶² The divergent roles of glutaminase inhibitors make this target an important point of study. Glutaminase overexpression is a critical factor in tumorigenesis and tumour progression and has been proposed to be a prognostic biomarker for various cancers, including ovarian cancer, osteosarcoma, hepatocellular carcinoma, colorectal cancer, and breast cancer.⁴⁵ Glutaminase inhibition in these tumours can induce two divergent roles, thereby resulting in an effective antitumor effect. Figure 3 illustrates various inhibitors identified as anticancer agents and their respective timelines of discovery.

Figure 3. Various GLS inhibitors and the respective timeline of their discoveries. The orthosteric or competitive inhibitors, which include DON, acivicin, JHU-083 and DRP-104, are mentioned in the red boxes. BPTES, CB-839, IACS-6274, Compound 13b and Compound 13, which belongs to the class of allosteric inhibitors, are mentioned in the pink boxes. Compound 968 which is a GLS2 selective molecule is mentioned in the blue box.

Cancer therapy and GLS modulators

• Orthosteric/competitive inhibitors

  Orthosteric/competitive inhibitors are also referred to as glutamine mimetics because they interact with the active site of the enzyme through covalent binding. Numerous potent orthosteric inhibitors have been developed, including DON, DRP-104, JHU-083, and acivicin (Figure 3). The glutamine mimetics DON and acivicin have been isolated from Streptomyces.²² DON and acivicin are glutamine antagonists that inhibit multiple glutamine/glutamate-binding enzymes, leading to severe toxicity in clinical trials due to their non-selective inhibition. To overcome DON toxicity, a prodrug strategy has been developed for efficient drug transfer to tumour tissues.⁶³ DRP-104, a newly synthesised DON prodrug, provided greater exposure to tumour tissues and exhibited a better tolerance profile.⁶⁴ Other DON prodrugs include JHU-083, Nedelcovych-13d, and Rais-5C.^22,65–67 Several crystal structures of human glutaminases with L-glutamate have been reported (PDBs: 3CZD, 3UO9, 3UNW, and 3SS5), which have aided in the development of these orthosteric inhibitors.

• Allosteric Inhibitors

  Unlike glutamine mimetics, this class of inhibitors selectively targets glutaminases without disrupting other aspects of glutamine metabolism. The allosteric site, located in the solvent region of the enzyme, is distinct from the catalytic site.²² Glutaminases exist in two forms: an inactive dimeric state and an active tetrameric state that triggers glutaminolysis.⁶⁸ The allosteric pocket, located at the interface of the GLS dimers, is hydrophobic and symmetric and allows symmetrical molecules, such as BPTES, to occupy this region²⁷ (Figure 4). Crystal structures (PDBs 3UO9, 5HL1, 3VP1, 3VOZ, 5JYO, and 4JKT) ^{28,37,68–70} have been used to study the allosteric binding pocket and its interactions, leading to extensive development of inhibitors at this site. It has been shown that the interaction of a ligand at the interface of the dimer triggers a significant conformational change at the key loop near the catalytic site, rendering the enzyme inactive.³⁷ BPTES is an early allosteric inhibitor that was developed to specifically target kidney-type glutaminases.⁷¹ In vitro studies have shown its high efficiency and specificity in inhibiting cancer cell proliferation; however, poor bioavailability and aqueous solubility prevent the use of BPTES.²² To overcome these limitations, modified BPTES derivatives have been developed. CB-839 and IACS-6274 (Figure 3) are more potent and orally bioavailable and have been tested in clinical trials.^32,72 In addition, IACS-6274 exhibits excellent pharmacokinetic and physicochemical properties and is currently undergoing phase I clinical trials for solid tumours.⁷²

Macrocycles have also been reported to inhibit GLS because they are designed based on existing crystal structures of GLS. Compound 13b shows an activity similar to that of the small molecule CB-839 on GLS.⁷³ Studied on ether-linker macrocycles as GLS inhibitors resulted in the discovery of compound 13.⁷⁴ Further studies on this category of inhibitors are required to identify potential therapeutic agents targeting GLS. Table 1 lists different GLS inhibitors and their current developmental stages.

Figure 4. Crystal structure of hKGA showing the allosteric and active sites. GLS forms a tetramer and the interface of dimers forms the allosteric site. Glutamine is the substrate for the active site and BPTES, a GLS inhibitor, occupies the allosteric site (Permission granted by the American Chemical Society from L. Chen & Cui, 2015).

Table 1. GLS inhibitors under development.

Drug Name	Company Name	Therapy Area	Indication	Development Stage
diazooxonorleucine (DON)	Kemmx Corp	Immunology	Rheumatoid Arthritis	Preclinical
Inhibitors of glutaminase (structure un-disclosed)	Cancer Research UK	Oncology	Unspecified Cancer	Preclinical
JHU-083	Dracen Pharmaceuticals Inc	Central Nerve System	Major Depressive Disorder	Preclinical
		Oncology	Colon Cancer; Lymphoma; Medulloblastoma; Melanoma	Preclinical
RP-10107 (structure un-disclosed)	Rhizen Pharmaceuticals SA	Oncology	Blood Cancer; Non-Small Cell Lung Cancer	Inactive
GLS inhibitor 13b	China Pharmaceutical University	Oncology	Triple-Negative Breast Cancer	Preclinical
telaglenastat hydrochloride (CB-839)	Calithera Biosciences Inc	Oncology	ALL; mCRPC; Melanoma; NHL; NSCLC; AML; MM; TNBC; mRCC; Anaplastic Astrocytoma; Cervical Cancer; GIST; Malignant Mesothelioma; Metastatic Colorectal Cancer; Myelodysplastic Syndrome; PDA; Neurofibrosarcoma; Soft Tissue Sarcoma	Discontinued
IPN-60090 (IACS-6274)	University of Texas MD Anderson Cancer Centre	Oncology	Anal Cancer; Endometrial Cancer; Gastric Cancer; Head and Neck Cancer Squamous Cell Carcinoma; Leiomyosarcoma; Lung Cancer; Melanoma; Ovarian Cancer; Solid Tumour	Phase 1

Resistance of glutaminase inhibitors

Despite the significant efficacy of GLS inhibitors, it has been observed that certain cancer cell lines eventually develop resistance to these inhibitors. Resistance is often attributed to compensatory pathways that allow tumour cells to evade glutaminase dependence. A study revealed that the treatment of pancreatic cells with a GLS inhibitor suppressed the proliferation of tumour cells in the early phase; however, the proliferation of these tumour cells continued both in vivo and in vitro. This change in the proliferation of these cell lines may be due to adaptive metabolic networks.⁷⁵ Notably, some altered pathways included the upregulation of supply pathways for the TCA cycle, which resulted in less dependence on glutamine.⁷⁶ Other pathways for glutaminase inhibition resistance include fatty acid oxidation (FAO) and mTORC1 signalling.⁷⁵ These compensatory pathways should be targeted for effective treatment with a glutaminase inhibitor. This has led to various combination therapy trials that showed a synergizing effect in different cancer models. Another study reported that combination therapy with GLS and PARP inhibitors could effectively treat chemo-resistant ovarian cancer.⁷⁷ Several target agents, such as erlotinib (EGFR inhibitor),⁷⁸ metformin (glycolysis inhibitor),⁷⁹ glutor (glucose uptake inhibitor),⁸⁰ and MLN128 (mTOR1 inhibitor),⁸¹ in combination with glutaminase inhibitors, have shown synergistic effects regarding activity. Figure 5 shows the inhibitors targeting multiple pathways that resulted in the reduction of the TCA cycle, thereby causing tumour suppression as the energy and building blocks required for growth were blocked.

Figure 5. Glutaminase inhibitor resistance through alternate metabolic pathways. Targeting glutaminase and alternate metabolic pathways prevent the TCA cycle, which results in the deprivation of energy and building blocks required for cell growth. Inhibitors are marked in red and the alternate pathways involved in resistance of GLS inhibitors is marked in purple. (Re-drawn with permission from Wang et al., 2020).

Mechanistic studies of CB-839 have indicated that myeloma cells undergo anti-proliferative effects due to caspase-dependent autophagy.⁸² A phase I clinical trial of CB-839 showed that CB-839 monotherapy was well tolerated and efficacious in a subset of patients,⁸³ and a combination therapy with CB-839 and standard chemotherapeutic agents was also studied in a phase I trial (NCT02071862) that showed encouraging clinical activity and tolerability. In a recent phase 1 clinical study (NCT05039801) of the IACS-6274 for the treatment of advanced solid tumours, IACS-6274 was found to have a half-life of 7.56 h, with plasma exposure showing a dose-dependent increase across dosage cohorts. IACS-6274 showed a robust pharmacokinetic and pharmacodynamic relationship across doses and patients (P < 0.001). DRP-104, an orthosteric inhibitor, is currently undergoing a phase 2 clinical trial for the treatment of solid tumours (NCT04471415). These findings suggest that GLS is a viable target for cancer therapy; however, more data are required to evaluate the efficacy of glutaminase inhibitors in clinical settings.

Cancer therapy and GLS2 modulators

GLS2, in contrast to GLS, has been shown to have a tumour-suppressive role. Nuclear translocation of GLS2 in cancer cells was shown to cause cell proliferation arrest and differentiation.⁸⁴ GLS2, a target gene of p53, suppresses tumour growth through the regulation of antioxidant function, resulting in different ROS levels in cells.⁸⁵ In contrast, overexpression of GLS2 resulted in tumour proliferation and metastasis in breast cancer.⁸⁶ GLS2 knockdown also sensitises cervical cancer cells to ionising radiation and reduces glutathione and NADH levels, indicating that GLS2 may be a therapeutic target in certain cancers.⁸⁷ Additionally, compound 968 was reported as a potential target for glutaminase activation in transformed cancer cells.⁸⁸ Subsequently, the sensitivity of compound 968 to GLS2 was studied, and it was demonstrated to have moderate selectivity for GLS2 over GLS.⁸⁹ Furthermore, alkyl benzoquinones inhibit mTORC1 and reduce cell proliferation. These compounds exhibit selectivity for GLS2 over GLS because of the presence of specific amino acid residues in the allosteric pocket.⁹⁰ In summary, the conflicting roles of GLS2 make it challenging to target it as an anticancer agent, and further investigation is needed to fully understand the role of GLS2 in different cancers.

Further applications

In addition to cancer, irregularities in glutamine metabolism are defining features of metabolic disorders, including obesity, diabetes, and non-alcoholic fatty liver disease.⁹¹ By modifying these metabolic pathways and decreasing the associated conditions, glutaminase inhibitors have the potential to offer a therapeutic approach for diverse metabolic disorders. By targeting glutamine metabolism, these inhibitors may aid in re-establishing the metabolic balance and improving patient outcomes.

In addition, disrupted glutamine metabolism has been linked to neurological disorders such as Alzheimer’s and Parkinson’s diseases.^92,93 It was demonstrated that neuronal excitotoxicity is caused by increased glutamate concentrations, indicating the need for glutaminase suppression in Alzheimer’s disease.⁹⁴ The increase in neuronal glutaminase activity is considered the primary reason for the upregulation of glutamate in the cerebral compartments during Parkinson’s disease,⁹⁵ indicating the need for therapeutic intervention targeting glutaminases in this disease. Glutaminase inhibitors offer a potential intervention strategy for these conditions by regulating glutamine levels and reducing neurotoxicity. However, the precise mechanisms and therapeutic benefits of these inhibitors in neurological diseases require further investigation.

Glutaminase inhibitors also hold promise for immunological regulation, presenting an exciting avenue due to their potential impact.⁹⁶ It was noted that glutamine metabolism in immune cells could undergo reprogramming, resulting in cells changing their phenotypes and functions.⁹⁷ These alterations in function and phenotype can contribute to pro- and anti-tumorigenic effects. These inhibitors may modulate immune responses by affecting glutamine-dependent immune cell function. This modulation may be beneficial in conditions characterised by autoimmune response and chronic inflammation, in which immunological dysregulation plays a significant role. A study demonstrated that blocking GLS using an inhibitor could potentially be a new treatment strategy for systematic lupus erythematosus, an autoimmune disease.⁹⁸

The applications of glutaminase inhibitors in medicine are highly promising and span multiple fields. From cancer treatment to metabolic disorders, neurological conditions, immune modulation, and organ transplantation, these inhibitors present a novel approach to tackling intricate health issues. Ongoing research and clinical trials will play a vital role in fully understanding and harnessing the capabilities of these inhibitors, ultimately paving the way for innovative therapies.

Discussion

Cancer is characterised by a metabolic shift that leads to uncontrolled cell proliferation and increased energy production and metabolite synthesis. Glutamine, an abundant amino acid in the human blood, plays a vital role in regulating biosynthesis and bioenergetic pathways in tumour growth. In addition, glutamine contributes to the production of antioxidants via glutaminolysis in tumour cells. Herein, we examine the role of glutaminolysis in cancer and the activity of glutaminase isoenzymes in this process. Pharmacological inhibition of glutaminases has shown significant activity against several cancers. Clinical trial molecules, including CB-839, IACS-6274, and DRP-104, suggest that inhibition of glutaminases can have a significant impact on tumour cells in terms of cell proliferation and apoptosis. Combination therapy with these inhibitors and other anticancer agents has also shown synergistic effects that can help overcome the resistance to glutaminase inhibitors. Identifying cancers with upregulated glutaminolysis and studying combination therapies are interesting methods for the treatment of certain cancers. The optimisation of glutaminase inhibitors with enhanced efficacy and favourable pharmacokinetic properties may represent a promising therapeutic strategy for inhibiting tumour growth. Combination therapy comprising glutaminase inhibitors and inhibitors targeting compensatory pathways may prove to be an effective treatment for cancers and other diseases that depend on glutamine. Recent studies have revealed the role of glutaminase in neural diseases and the need for glutaminase inhibitors with good permeability through the blood-brain barrier has become a necessity. The multiple roles of glutaminase make it an interesting target for further study, and the development of glutaminase inhibitors with enhanced efficacy and favourable pharmacokinetic properties can play an important role in the treatment of different diseases.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by the Korea Drug Development Fund funded by the Ministry of Science and ICT; the Ministry of Trade, Industry, and Energy; the Ministry of Health and Welfare [HN21C1180]; and the Korea Research Institute of Chemical Technology [KK2331-20]. The authors thank Editage (www.editage.co.kr) for English language editing.