New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer.

BACKGROUND: Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients. METHODS: A panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity. RESULTS: The anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness. CONCLUSION: This study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism.

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 molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.

GAC inhibitors with a 4-hydroxypiperidine spacer: Requirements for potency.

Allosteric inhibitors of glutaminase (GAC), such as BPTES, CB-839 and UPGL00019, have great promise as inhibitors of cancer cell growth, but potent inhibitors with drug-like qualities have been difficult to achieve. Here, a small library of GAC inhibitors based on the UPGL00019 core is described. This set of derivatives was designed to assess if one or both of the phenylacetyl groups flanking the UPGL00019 core can be replaced by smaller simple aliphatic acyl groups without loss in potency. We found that one of the phenylacetyl moieties can be replaced by a set of small aliphatic moieties without loss in potency. We also found that enzymatic potency co-varies with the VDW volume or the maximum projection area of the groups used as replacements of the phenylacetyl moiety and used literature X-ray data to provide an explanation for this finding.

Mechanistic Basis of Glutaminase Activation: A KEY ENZYME THAT PROMOTES GLUTAMINE METABOLISM IN CANCER CELLS.

Glutamine-derived carbon becomes available for anabolic biosynthesis in cancer cells via the hydrolysis of glutamine to glutamate, as catalyzed by GAC, a splice variant of kidney-type glutaminase (GLS). Thus, there is significant interest in understanding the regulation of GAC activity, with the suggestion being that higher order oligomerization is required for its activation. We used x-ray crystallography, together with site-directed mutagenesis, to determine the minimal enzymatic unit capable of robust catalytic activity. Mutagenesis of the helical interface between the two pairs of dimers comprising a GAC tetramer yielded a non-active, GAC dimer whose x-ray structure displays a stationary loop ("activation loop") essential for coupling the binding of allosteric activators like inorganic phosphate to catalytic activity. Further mutagenesis that removed constraints on the activation loop yielded a constitutively active dimer, providing clues regarding how the activation loop communicates with the active site, as well as with a peptide segment that serves as a "lid" to close off the active site following substrate binding. Our studies show that the formation of large GAC oligomers is not a pre-requisite for full enzymatic activity. They also offer a mechanism by which the binding of activators like inorganic phosphate enables the activation loop to communicate with the active site to ensure maximal rates of catalysis, and promotes the opening of the lid to achieve optimal product release. Moreover, these findings provide new insights into how other regulatory events might induce GAC activation within cancer cells.

Design and evaluation of novel glutaminase inhibitors.

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.

Simultaneously targeting tissue transglutaminase and kidney type glutaminase sensitizes cancer cells to acid toxicity and offers new opportunities for therapeutic intervention.

Most cancer cells undergo characteristic metabolic changes that are commonly referred to as the Warburg effect, with one of the hallmarks being a dramatic increase in the rate of lactic acid fermentation. This leads to the production of protons, which in turn acidifies the microenvironment surrounding tumors. Cancer cells have acquired resistance to acid toxicity, allowing them to survive and grow under these detrimental conditions. Kidney type glutaminase (GLS1), which is responsible for the conversion of glutamine to glutamate, produces ammonia as part of its catalytic activities and has been shown to modulate cellular acidity. In this study, we show that tissue, or type 2, transglutaminase (TG2), a γ-glutamyl transferase that is highly expressed in metastatic cancers and produces ammonia as a byproduct of its catalytic activity, is up-regulated by decreases in cellular pH and helps protect cells from acid-induced cell death. Since both TG2 and GLS1 can similarly function to protect cancer cells, we then proceeded to demonstrate that treatment of a variety of cancer cell types with inhibitors of each of these proteins results in synthetic lethality. The combination doses of the inhibitors induce cell death, while individual treatment with each compound shows little or no ability to kill cells. These results suggest that combination drug treatments that simultaneously target TG2 and GLS1 might provide an effective strategy for killing cancer cells.

Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death.

Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death.

Transglutaminase 2 (TG2) is a GTP-binding/protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that maintain the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotide-bound TG2 adopts a monomeric closed conformation while calcium-bound TG2 assumes an open conformational state that can form higher order oligomers. SAXS analysis also suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time-resolved SAXS to show that LM11 increases the ability of calcium to drive TG2 to an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Opportunities for targeted therapies: trametinib as a therapeutic approach to canine oral squamous cell carcinomas.

Oral tumors are relatively common in dogs, and canine oral squamous cell carcinoma (COSCC) is the most prevalent oral malignancy of epithelial origin. COSCC is locally aggressive with up to 20% of patients showing regional or distant metastasis at the time of diagnosis. The treatment of choice most typically involves wide surgical excision. Although long-term remission is possible, treatments are associated with significant morbidity and can negatively impact functionality and quality of life. OSCCs have significant upregulation of the RAS-RAF-MEK-MAPK signaling axis, and we had previously hypothesized that small-molecule inhibitors that target RAS signaling might effectively inhibit tumor growth and progression. Here, we demonstrate that the MEK inhibitor trametinib, an FDA-approved drug for human cancers, significantly blocks the growth of several COSCC cell lines established from current patient tumor samples. We further show clinical evidence that the drug is able to cause significant tumor regression in some patients with spontaneously occurring COSCC. Given the limited treatment options available and the high rate of owner rejection of these offered options, these findings provide new hope that more acceptable treatment options may soon enter the veterinary clinic.

Alone and together: current approaches to targeting glutaminase enzymes as part of anti-cancer therapies.

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.

Many strategies exist to inhibit cancer progression, from chemotherapy to more targeted therapies that exploit differences between tumors and healthy tissue. One such targeted strategy involves inhibition of the enzyme glutaminase, which converts glutamine obtained from the bloodstream into nutrients that fuel tumor growth. Research into glutaminase is ongoing, with regulation of the enzyme, and novel molecular approaches to inhibit its activity, being key focus areas. Here, we review recent progress on targeting glutaminase enzymes for anti-cancer therapy, including several approaches in which glutaminase inhibitors are combined with inhibitors of other cancer-relevant targets, to increase the overall effectiveness of the treatment.

Confirmation of canine acanthomatous ameloblastoma using RAS Q61R immunohistochemical staining of formalin-fixed paraffin-embedded tissues.

Differentiating canine acanthomatous ameloblastoma (CAA) from oral squamous cell carcinoma (OSCC) based on routine histopathology can be challenging. We have previously shown that more than 95% of CAAs harbor an HRAS p.Q61R somatic mutation, while OSCCs carry either wild-type alleles or other MAPK pathway activating mutations (e.g., HRAS p.Q61L, BRAF p.V595E). Given that HRAS p.Q61R mutations are highly prevalent in CAA, we hypothesized that a RAS Q61R-specific rabbit monoclonal antibody may be a useful tool for confirmation of CAA by immunohistochemical (IHC) staining. In the present study, we assessed IHC staining of archived formalin-fixed and paraffin-embedded biopsy samples with a diagnosis of CAA (n = 23), using a RAS Q61R-specific rabbit monoclonal antibody (SP174) and an automated IHC stainer. Negative control samples consisted of HRAS p.Q61R mutation-negative OSCC tumors with either a known HRAS p.Q61L mutation (n = 1), BRAF p.V595E mutation (n = 4), or wild-type corresponding alleles (n = 3). We found that all 23 CAAs showed diffuse and strong membranous RAS Q61R immunoreactivity (100% sensitivity), while none of the 8 OSCCs showed immunoreactivity (100% specificity). The data supports the use of RAS Q61R-specific rabbit monoclonal antibody for diagnostic IHC confirmation of CAA and ruling out OSCC in dogs.

Highly recurrent BRAF p.V595E mutation in canine papillary oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common oral epithelial malignancy in dogs. It exhibits locally aggressive biological behaviour with the potential to metastasize, and a reported 1-year survival rate of 0% when left untreated. Expression studies suggest that aberrant MAPK signalling plays a key role in canine OSCC tumorigenesis, which is consistent with BRAF and HRAS MAPK-activating mutations reported in some tumours. Several morphological subtypes of canine OSCC have been described, with papillary, conventional, and basaloid as the most common patterns. We hypothesized that mutational differences may underlie these phenotypic variations. In this study, targeted Sanger sequencing and restriction fragment length polymorphism assays demonstrate that up to 85.7% of canine papillary OSCC (n = 14) harbour a BRAF p.V595E mutation. Assessment of neoplastic epithelial cell proliferation using Ki67 immunolabelling (n = 10) confirmed a relatively high proliferation activity, consistent with their known aggressive clinical behaviour. These findings underscore a consistent genetic feature of canine papillary OSCC and provide a basis for the development of novel diagnostic and targeted therapeutic approaches that can improve the quality of veterinary care.

Inhibiting Glutamine Metabolism Blocks Coronavirus Replication in Mammalian Cells.

Developing therapeutic strategies against COVID-19 has gained widespread interest given the likelihood that new viral variants will continue to emerge. Here we describe one potential therapeutic strategy which involves targeting members of the glutaminase family of mitochondrial metabolic enzymes (GLS and GLS2), which catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. We show three examples where GLS expression increases during coronavirus infection of host cells, and another in which GLS2 is upregulated. The viruses hijack the metabolic machinery responsible for glutamine metabolism to generate the building blocks for biosynthetic processes and satisfy the bioenergetic requirements demanded by the 'glutamine addiction' of virus-infected host cells. We demonstrate how genetic silencing of glutaminase enzymes reduces coronavirus infection and that newer members of two classes of small molecule allosteric inhibitors targeting these enzymes, designated as SU1, a pan-GLS/GLS2 inhibitor, and UP4, which is specific for GLS, block viral replication in mammalian epithelial cells. Overall, these findings highlight the importance of glutamine metabolism for coronavirus replication in human cells and show that glutaminase inhibitors can block coronavirus infection and thereby may represent a novel class of anti-viral drug candidates. Teaser: Inhibitors targeting glutaminase enzymes block coronavirus replication and may represent a new class of anti-viral drugs.

Exploring the Role of Transglutaminase in Patients with Glioblastoma: Current Perspectives.

Tissue transglutaminase (tTG) is a rather unique GTP-binding/protein crosslinking enzyme that has been shown to play important roles in a number of cellular processes that impact both normal physiology and disease states. This is especially the case in the context of aggressive brain tumors, such as glioblastoma. The diverse roles played by tTG in cancer survival and progression have led to significant interest in recent years in using tTG as a therapeutic target. In this review, we provide a brief overview of the transglutaminase family, and then discuss the primary biochemical activities exhibited by tTG with an emphasis on the role it plays in glioblastoma progression. Finally, we consider current approaches to target tTG which might eventually have clinical impact.

Comparative transcriptional profiling of canine acanthomatous ameloblastoma and homology with human ameloblastoma.

Ameloblastomas are odontogenic tumors that are rare in people but have a relatively high prevalence in dogs. Because canine acanthomatous ameloblastomas (CAA) have clinicopathologic and molecular features in common with human ameloblastomas (AM), spontaneous CAA can serve as a useful translational model of disease. However, the molecular basis of CAA and how it compares to AM are incompletely understood. In this study, we compared the global genomic expression profile of CAA with AM and evaluated its dental origin by using a bulk RNA-seq approach. For these studies, healthy gingiva and canine oral squamous cell carcinoma served as controls. We found that aberrant RAS signaling, and activation of the epithelial-to-mesenchymal transition cellular program are involved in the pathogenesis of CAA, and that CAA is enriched with genes known to be upregulated in AM including those expressed during the early stages of tooth development, suggesting a high level of molecular homology. These results support the model that domestic dogs with spontaneous CAA have potential for pre-clinical assessment of targeted therapeutic modalities against AM.

Patents targeting the Warburg effect for cancer therapy: an interview with William P Katt.

Dr William Katt is a multidisciplinary scientist with particular focus in computational, synthetic and biological chemistry. He obtained his undergraduate degree at Rensselaer Polytechnic Institute and performed his graduate studies at Yale University, where he focused on designing small-molecule inhibitors of the Rho/Rho GEF interaction. Following those studies, Dr Katt accepted a fellowship from the American Cancer Society which funded his work at Cornell University, where he investigated small-molecule inhibitors of the enzyme glutaminase, a key player in cancer metabolism. Today, Dr Katt is a research associate at Cornell and maintains a number of collaborations with researchers across the nation examining glutaminase, cancer stem cells, nano-therapeutics and more, with the goal of developing therapeutic approaches that will eventually help patients in the clinic.

Inhibition of cancer metabolism: a patent landscape.

Cancer metabolism is currently a hot topic. Since it was first realized that cancer cells rely upon an altered metabolic program to sustain their rapid proliferation, the enzymes that support those metabolic changes have appeared to be good targets for pharmacological intervention. Here, we discuss efforts pertaining to targets in cancer metabolism, focusing upon the tricarboxylic acid cycle and the mechanisms which feed nutrients into it. We describe a broad landscape of small-molecule inhibitors, targeting a dozen different proteins, each implicated in cancer progression. We hope that this will serve as a reference both to the areas being most highly examined today and, relatedly, the areas that are still ripe for novel intervention.

Starving the Devourer: Cutting Cancer Off from Its Favorite Foods.

Increased consumption of glucose and glutamine are metabolic hallmarks of tumorigenesis. In this issue of Cell Chemical Biology, Reckzeh et al. (2019) describe the discovery of Glutor, a potent inhibitor of cellular glucose uptake. Combining Glutor with the glutaminase inhibitor CB-839 creates a metabolic crisis in cancer cells, synergistically impeding proliferation.