Overview of the Development of Glutaminase Inhibitors: Achievements

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Perspective

Overview of the development of Glutaminase Inhibitors: Achievements and Future Directions Xi Xu, Ying Meng, Lei Li, Pengfei Xu, Jubo Wang, Zhiyu Li, and Jinlei Bian J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00961 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Journal of Medicinal Chemistry

Overview of the development of Glutaminase Inhibitors: Achievements and Future Directions Xi Xu,† Ying Meng, † Lei Li, † Pengfei Xu, † Jubo Wang, † Zhiyu Li, *,†,‡ Jinlei Bian*,†,‡ †

Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing

210009, P.R.China ‡

Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,

Nanjing 21009, China * Corresponding author. Tel.: +86 13951678592 (Z. Li); +8615151865295 (J. Bian) E-mail address: [email protected] (Z. Li); [email protected] (J. Bian) Abstract: It has been demonstrated that glutamine metabolism has become the main energy and building blocks supply for the growth and viability of a potentially large subset of malignant tumors. The glutamine metabolism often depends upon mitochondrial glutaminase (GLS) activity, which converts glutamine to glutamate and serves as a significant role for bioenergetic processes. Thus, recently, the GLS has become a key target for small molecule therapeutic intervention. Numerous medicinal chemistry studies are currently aimed at the design of novel and potent inhibitors for GLS, however, to date, only one compound (named CB-839) have entered clinical trials for the treatment of advanced solid tumors and hematological malignancies. The perspective summarizes the progress in the discovery and development of GLS inhibitors, including the potential binding site, biochemical techniques for inhibitor identification and approaches for identifying small-molecule inhibitors, as well as future therapeutic perspectives in glutamine metabolism are also put forward in order to provide reference and rational for the drug discovery of novel and potent glutamine metabolism modulators.

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1. INTRODUCTION 1.1. An Overview of the Glutamine Metabolic Pathway and Related Catalytic Enzymes Cancer cells develop extensive reprogramming of cellular energy metabolism.1 Tumor cells are different from normal cells in their utilization of glucose which are more likely to convert glucose to lactic acid by glycolysis and transport it out of the cell, even under conditions of sufficient oxygen. This phenomenon was called the Warburg effect which first proposed nearly a century ago.2 In addition to glucose metabolism, the involvement of glutamine metabolism has attracted attention as a potential hallmark for the development of novel therapeutic drugs for cancer treatment.3 Glutamine (Gln) is the most abundant amino acid in plasma4 and serves as a significant role in many energy-generating and biosynthesis process for the growth and proliferation of cancer cells by entering the tricarboxylic acid (TCA) cycle.3 Under catabolic stressed conditions e.g., post-operation, injury or sepsis, glutamine is dramatically consumed by kidney, gastrointestinal tract and immune compartment.116 Studies have demonstrated that the intestinal mucosa cells particularly dependent on glutamine undergo necrosis if glutamine is deprived.116 Moreover, some growing cancer cells also exhibited highly dependence on glutamine, with rapidly dying after glutamine depletion.117,118 This so-called “glutamine addiction” has been well characterized in cancers including glioblastoma (GBM), leukemia, lymphomas, lung, triple-negative breast cancer and pancreatic cancer.5-10 Glutamine enters into cells via transporter or macrocytosis, e.g., the solute carrier family 1 neutral amino acid transporter member 5 (SLC1A5, also known as ASCT2), which is one of transporter proteins.11 After glutamine enters the cell, a significant fraction of it is converted to glutamate (Glu) and ammonia by glutaminase (GLS) in the mitochondria.12,13 Glu is further oxidatively deaminated into α-ketoglutarate (α-KG)


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through two mechanisms.14 The two mechanism involved two key enzyme, one of which is aminotransferases involved in maintaining the stability of intracellular amino acid pools to provide non-essential amino acids for cell growth.15-18 The other is glutamate dehydrogenase (GDH), which is widely expressed in the liver and catalyzes the reversible deamination of glutamate to produce α-KG and ammonium at near thermodynamic equilibrium.14,19 This α-KG then enters the tricarboxylic acid (TCA) cycle and involves in the production of nucleotides, ATP, certain amino acids, lipids and glutathione in mitochondria (Figure 1).20 Thus, Glutamine can fulfill both the energetic and biomass requirements of proliferating cells in this way. Throughout the metabolic pathways, the requirement is met by the overexpression of GLS, which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target.

Figure 1. An overview of the glutamine metabolic pathway for therapy in cancer. Glutamine is imported via SLC1A5 (also known as ASCT2).11 Glutamine can also be exchanged for essential amino acids, such as leucine through the L-type amino acid transporter 1 (LAT1).112 Glutamine



can also contribute to nucleotide biosynthesis and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis for support of protein folding and trafficking.113 Glutamine is hydrolyzed to glutamate through glutaminase (GLS1/2). Glutamate can be converted to α-ketoglutarate (α-KG) through glutamate dehydrogenase (GDH) or aminotransferases.14 Through the actions of various aminotransferases, e.g., phosphoserine aminotransferase 1 (PSAT1), glutamic pyruvate transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), glutamate derived from glutamine can supply nitrogen atoms for the biosynthesis of amino acids. In addition, glutamine can also directly provide nitrogen for asparagine by asparagine synthetase (ASNS). α-KG enters the tricarboxylic acid (TCA) cycle and can provide energy and building blocks for the growth and proliferation of cell. Modulators that target various aspects of glutamine metabolism are shown in red. 1.2. The First Step in the Glutaminolysis: Phosphate-Activated Glutaminase (GLS) Glutaminase has been proved to control the first step in the glutaminolysis pathway which has become an intriguing and promising target for the developing antitumor drugs. By now, four informs of human glutaminase have been identified, including kidney-type glutaminase (KGA/GLS1), the splice KGA variant (Glutaminase C or GAC), Liver-type glutaminase (LGA/GLS2) and glutaminase B (GAB).21,22 The transcripts of KGA and GAC belong to GLS (GLS1) gene, while LGA and GAB for GLS2. GLS gene is overexpressed in many tumor cell lines and primary tumors while GLS2 gene is not widely expressed in tumors.23 Treating with efficient GLS1 inhibitor in different tumor models, or by genetic silencing of GLS1 have validated GLS1 as a therapeutic target.26,35,38,114,115 Expression of GAC, which is more active than KGA, is increased in a number of cancers, indicating that GLS alternative splicing may have an important role in the


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presumed higher glutaminolytic flux in cancers.27-32 As for GLS2, it showed more complex roles in cancers. Upregulated GLS2 enzymatic activity has also been demonstrated to be related with tumor cell growth in vitro and in vivo recently33 though there is controversy over the role of GLS2 as a tumor suppressor.34 The context-dependent role of GLS2 in cancer need further study and validation. It is worth noting that GLS2 is mainly found in adult liver, while GLS1 is widely expressed throughout extra-hepatic tissues which is considered to be a definite target for tumor suppression.35 In addition, GLS1 has recently been intensively studied, as it has been linked to the progression of various cancers.36 The evaluated activity of GLS1 has been correlated with a number of pathways, such as HIF1α, cMyc, Raf, miRNA23, EGFR and Ras/MAPK, as well as to the hyper-activation of Rho GTpases.37-43 Thus, GLS1 would be a potentially more attractive target for inhibiting cancer cell growth compared with GLS2. To data, there is no compound has been progressed as a potential GLS1 inhibitor from discovery to market. Indeed, most of these modulators are still in need of substantial medicinal chemistry optimization. In this review, we focused on the discovery and development of small molecule modulators targeting glutaminase. In addition, the molecular basis and clinical progress of the representative compounds are also reviewed. Future directions and potential challenges faced in the race to develop new therapeutics in this field are discussed to provide a reference for developing novel glutaminase modulator for the treatment of cancers. 2. GLS1 STRUCTURAL ASPECTS The two splicing variants KGA and GAC, which derived from GLS1 genes, share a common N-terminal sequence (1-550) but contain unique C-terminal regions (551-669 for KGA, 551-598 for GAC).44 Generally, GLS1 exists as either a dimer or a tetramer. The dimer is inactive while the



tetramer showed catalytic activity.13 It has been shown that the dimer to tetramer transition is necessary for the enzymatic activation of GLS1. The activated tetramer could be facilitated in vitro based on the addition of inorganic phosphate or other polyvalent anions, and substrate entry to the pocket by competing with the product glutamate.45 The activity of KGA or GAC is also modulated by several compounds, e.g., glutamate, citrate, calcium, protons, fatty acyl-CoA derivatives, TCA cycle intermediates and certain long chain fatty acids under physiological conditions.33,119 The acyl-CoA derivatives have dual effects on phosphate-activated GLS with low concentrations activate the enzyme, but inhibit at higher concentrations. Generally, acyl-CoA derivatives are more effective inhibitors when the fatty acyl chain is elongated and more effective activators on unsaturation of the fatty acyl group.119 Recently, the structure of GLS1 has been determined by X-ray crystallography (Table 1), revealing the presence of four molecules in the asymmetric unit (Figure 2). This quaternary involves two sets of interfaces and one interface is consisted of the contacts between monomers a and b, c and d. The other interface is made up of the contacts between monomers a and c, b and d. The first interface buries considerably more surface area than the second interface. The constructs employed for crystallography include the isolated glutaminase catalytic domain of KGA and the entire biologically processed form of the enzyme (refer to GAC, residues 71-598). The Ile221-Leu533 has been considered as the catalytic domain of KGA (Figure 2A). Neither the N- nor C-termini of GLS1 has been resolved up to now. However, the C-terminus has been proved to influence the enzyme function due to GAC has greater catalytic activity than KGA. Recently, structures with substrate glutamine, glutamate or assorted inhibitors have also been determined (e.g., PDB ID: 3UO9, 4O7D and 5JYO).47,49,52 According to the information of these crystal structures, we found that there were different binding


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sites for ligand occupying to inhibit the enzyme activity of GLS1. Two main pockets which have been confirmed by X-ray crystallography are substrate binding pocket and allosteric binding pocket (Figure 2B).

Figure 2. Schematic view and structure of GLS. (A) KGA/GAC domains. (B) Structure and binding sites of GLS1. Allosteric binding pocket is shown in red, and substrate catalytic site is shown in green.

Table 1. Summary of the Available Crystal Structures of GLS1 and GLS1-ligand Complexa Residues GLS1 isoform

Ligand

PDB code

Publication year

used Apo GLS1 GAC

72-598

None

5D3O

2015.08.06

GLS

221-533

None

3VOY46

2012.02.23



GAC (m)b

134-609

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None

3SS328

2011.07.07

GLS1 with substrates or phosphate GLS1

221-533

L-glutamine

3VP046

2012.02.23

GAC (m) b

134-609

Phosphate

3SS428

2011.07.07

GAC (m) b

134-609

L-glutamate

3SS528

2011.07.07

GAC

71-598

L-glutamate

3UNW47

2011.11.16

GLS1

221-533

L-glutamate

3CZD46

2008.04.29

GLS1 with inhibitors GAC

60-598

UPGL00004

5WJ648

2017.07.21

GLS1

139-656

BPTES

5UQE

2017.02.08

GLS1

221-533

CB-839

5JYO49

2016.05.15

GLS1

221-533

Trans-CBTBP

5JYP49

2016.05.15

GAC

72-598

UPGL_00019

5I9450

2016.02.19

GAC

72-598

CB-839

5HL1

2016.01.14

GAC

72-598

UPGL_00009

5FI250

2015.12.22

GAC

72-598

UPGL_00011

5FI650

2015.12.22

GAC

72-598

UPGL_00015

5FI750

2015.12.22

GLS1

221-531

DON

4O7D52

2013.12.24

GAC (m) b

128-555

BPTES

4JKT51

2013.03.11

GLS1

221-533

BPTES

3VOZ46

2012.02.23

GLS1

221-533

BPTES

3VP146

2012.02.23

GLS1

221-533

BPTES derivate 2

3VP246

2012.02.23

GLS1

221-533

BPTES derivate 3

3VP346

2012.02.23

GLS1

221-533

BPTES derivate 4

3VP446

2012.02.23

GAC

71-598

BPTES

3UO947

2011.11.16

a

The table is an update and specific classification of the table 3 form Katt et al23. bm represents

mouse.


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2.1 Substrate Catalytic Site Until 2008, the structural features of the enzyme were disclosed for the first time by ʟ-glutamate-bound structure (PDB code: 3CZD).46 After that, several other crystal structures containing ʟ-glutamate have also been reported (PDB code: 3UO9, 3UNW, 3SS5, and 3VP1). The structure of GLS1 has two domains with the catalytic site located at the interfaces. The domain I consists of Ile221-Pro281 and Cys424-Leu533 of a five-stranded anti-parallel β-sheet surrounded by six α-helices and several loops (Figure 3A, marked as orange). The domain II comprises Phe282-Thr423 of seven α-helices (Figure 3A, marked as blue). The active site is highly basic and the substrate makes several key hydrogen-bonding interactions and hydrophobic interactions with Tyr249, Gln285, Ser286, Asn335, Glu381, Asn388, Tyr414, Tyr466, and Val484 (Figure 3B). The distance between the side chain of glutamate and Ser286 is within hydrogen-bonding range, while other key residues Lys289, Tyr414 and Tyr466 are in the vicinity of the active site. The carbonyl oxygen of the substrate glutamine could form hydrogen-bond with the main chain amino groups of Ser286 and Val484, composing the oxyanion hole.47 In addition, Lys289 and Tyr466 could form hydrogen-bond with Ser286, while Lys289 acts as a general base for the nucleophilic attack by accepting the proton from Ser286, Tyr466 is involved in proton transfer during catalysis.



Figure 3. Structure and important amino acids of substrate catalytic site and allosteric binding pocket of GLS1. (A) Structure of GLS1 and bound substrate (GLN) is shown as a purple stick. N-terminal is labeled as N and C-terminal is labeled as C. (B) The key residues of substrate catalytic site and hydrogen bonds with substrate GLN are shown. The hydrogen bonds were shown as dotted lines. (C) The allosteric binding pocket of GLS1. (D) The key residues in the allosteric binding pocket. 2.2 Allosteric Binding Pocket Except for the catalytic site, there is an allosteric pocket in the solvent-exposed region at the dimer interface of GLS1 for ligand to occupy to inhibit the protein activity. Recently, several crystal structures (PDB ID: 3UO9, 3VP1, 3VOZ, 5JYO and 4JKT)46,47,49,51 have disclosed the allosteric binding pocket, opening the way for structure-based design of GLS1 inhibitors. The allosteric binding pocket located about 18 Å away from the active site Ser286 (Figure 3C). The


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hydrophobic pocket consists of Phe318, Leu321, Phe322, Leu323, Asn324, Glu325 and Tyr394 from each monomer and the side chain of Phe322 is found at the bottom of the allosteric pocket (Figure 3D). When ligand occupies the allosteric pocket, it would interact with loop region comprised of residues 312-329 and interface helix α-13, residues 386-399, triggering a dramatic conformational change of the key loop (Leu316 to Leu320) referred to as the “gating loop or activation loop” near the catalytic site and rendering it inactive.28,51 Therefore, by binding in an allosteric pocket, ligands would inhibit the enzymatic activity through triggering a major conformational change on the key residues which involves in stabilizing the active sites and regulating its enzymatic activity. In sum, a stable inactive tetrameric GLS1 would be formed through occupying the allosteric pocket by small molecule modulators. Some other binding pockets have also been speculated which could be occupied by small molecules to inhibit the enzyme activity according to molecular modeling and site-directed mutagenesis studies. We would present them combined with specific inhibitors in the following paragraphs. 3. Assays to Monitor GLS1 Modulation It is significant to study the assay technology platforms for discovering GLS1 inhibitors due to the potential mechanism of compound action and compound-mediated assay interference. Such knowledge would be helpful for the discovery of efficacious GLS1 inhibitors. Therefore, we will describe currently available assays for the identification and evaluation of GLS1 inhibitors. Basic principles of the different assay types will be included. 3.1 Enzymatic Assays Understanding the underlying biochemical principles of GLS1 assays is necessary to interpret



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apparent compound response. Based on the assay readout, certain types of compounds mediated interferences may be enriched while others are negligible. Many parameters can profoundly affect observed compound activity, including buffer composition and reaction time (Table 2). The mainly principle of enzymatic assays for discovering and evaluating GLS1 inhibitors is the hydrolysis of glutamine to glutamate. 3.1.1 Glutamine Hydrolysis Assay

Two-step glutaminase assay.53 GLS1 can catalyze the hydrolysis of glutamine to glutamate which can be further converted to α-KG and NADH/NADPH through the oxidative deamination of glutamate dehydrogenase. In the process, hydrazine is used to react with α-KG to form hydrazide in order to completely oxidize the glutamate. The activity of glutaminase can be quantitatively reflected by measuring the absorbance of NADH/NADPH at 340 nm. The disadvantage of the method is the relatively short and unspecific wavelengths at which many other organic compounds absorb (Table 3). Therefore, as an alternative, a three-step enzyme assay system is used to evaluate the inhibitory activity. Table 2. Composition of Enzymatic Assay Class

Reagent

Concentration

protein substrate

glutaminase glutamine potassium phosphate EDTA Tris-acetrate, pH 8.6

0.1 mM 20 mM 150 mM 0.2 mM 50 mM

HCl

3N

The first step solvent

stop solution protein The second step

substrate solvent

glutamate dehydrogenase NAD+/NADP+ Tris-acetrate, pH 9.4 hydrazine

Comment

inactivate the glutaminase

0.4 mg/mL 2 mM 80 mM 0.2 M


reacting with α-KG

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ADP

0.25 mM

Three-step glutaminase assay.54 Similar to the two-step glutaminase assay, the method also employed the principle that glutamine can be hydrolyzed to glutamate. Firstly, glutamine is hydrolyzed to glutamate and ammonia. Then, through the oxidation of glutamate oxidase, glutamate is converted to α-KG, ammonia and hydrogen peroxide (H2O2). Finally, the H2O2 is reacted with Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) in a 1:1 stoichiometry in the presence of horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin. Based on resorufin fluorescence (excitation at 530-560 nm, emission detection at 590 nm), the inhibition activity of various compounds against glutaminase can be quantitatively measured. The advantage of this method is that the autofluorescence of most biological samples has little interference with the emission fluorescence of resorufin (Table 3). In addition, in order to repeat the second-step reaction and amplify the detection signal, L-alanine and L-glutamate-pyruvate transaminase can be included to regenerate glutamate by transamination of α-KG. With the exception of glutaminase, all other enzymes and reagents mentioned above can be found in Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen, Cat. no. A12221). Several researchers also made use of the kit to detect the level of intracellular glutamate so as to reflect the inhibitory activity of compounds against glutaminase.26

Radiolabeled Glutamine Assay.54 Except for the methods that determine the hydrolyzed product, the radiolabeled glutamine assay can also determine the enzymatic activity in a direct way. The method employed radiolabeled glutamine, [3H]-glutamine as substrate to determine the activity of glutaminase inhibitors. After incubation of [3H]-glutamine, inhibitors and glutaminase together, the substrate and reaction product are then isolated through 96-well spin columns packed



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with strong anion ion-exchange resin. Imidazole buffer is used to wash the unreacted [3H]-glutamine. Then the reaction product, [3H]-glutamate, which is eluted with HCl and analyzed for radioactivity so as to reflect the inhibitory potency of inhibitors in glutaminase. Comparing these three methods, the two-step or three-step glutaminase assay using the principle of quantifying glutamate by coupling to glutamate dehydrogenase-mediated NADH production are the most commonly method used to measure the inhibitory activity of GLS1. While the radioactive assay has the advantage of producing fewer false positives due to assay interference compared with the other two methods (Table 3) Table 3. Summary of Glutamine Hydrolysis Assays for Evaluating GLS1 Inhibitors Assay Two-step glutaminase assay

Advantages

Three-step glutaminase assay

Basic instrumentation requirements Continuous readout option Relative higher throughput Basic instrumentation requirements Continuous readout option Relative higher throughput

Disadvantages

Short wavelength readout High enzyme concentrations required

Greater complexity than two-step method

Radiolabel glutaminase assay

High analytical sensitivity More reliable

End-point readout Analyte separation step required Radiation safety and disposal requirements Specialized instrumentation requirements

3.1.2 GLS1 Binding Assay The most straightforward parameter reflecting binding strength of compounds is the equilibrium dissociation constant Kd. Hence, assays that can determine Kd and precisely measure direct binding affinity are valuable in evaluating GLS1 inhibitors. However, these methods have not been widely used to screen for novel GLS1 inhibitors compared with the glutamine hydrolysis


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assays. The combination of glutamine hydrolysis assays and binding assays are supported to ensure the precise assessment of GLS1 inhibitors and provide useful information for further optimization.

Fluorescence resonance energy transfer (FRET)-based assay. As for allosteric inhibitors, some proximity-based fluorescent methods have also been developed. Among them, a fluorescence resonance energy transfer (FRET)-based assay has been established recently.105 FRET is the radiation-free transmission of energy from a donor molecule that initially absorbs the energy to an acceptor molecule to which the energy is subsequently transferred. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and accepter.106 The character makes the FRET method sensitive to small changes in distance which can be adapted to detect the distance of dimer-to-tetramer transition. Cerione group has successfully developed a FRET assay to monitor the effects that allosteric inhibitors have on GAC tetramer formation in real time.105 Addition of the allosteric inhibitors to GAC labeled with FRET pairs increased the FRET readout, indicating the rapid formation of tetramers upon the binding of the inhibitors. The method has also been employed to evaluate the mechanism of GAC activation and illustrate how a distinct class of allosteric inhibitors impacts the metabolic program of transformed cells.

Bio-layer interferometry (BLI)-based assay.107 The equilibrium dissociation constant Kd is the most straightforward parameter which reflect binding strength. Thus, assays which can determine Kd and precisely measure direct binding are useful in evaluating GLS1 inhibitors. BLI is a commonly used technique for the binding kinetic method and can monitor the association and dissociation process in real time and obtain the association rate constant kon and the dissociation



rate constant koff. Ruan et al. developed a direct kinetic binding assay for KGA using BLI as a detection method for rigorous characterization. The biomolecular interaction analysis of compounds binding to the biotinylated KGA protein was performed using a ForteBio K2 instrument with Super Streptavidin (SSA) biosensors. Human KGA showed does dependent direct binding to its substrate Gln (Kd 4 µM) and the allosteric inhibitor BPTES (Kd 0.2 µM). In the BLI-based assays, the small molecule would be identified as a potent and strong binder if Kd＜100 nM. The developed BLI-based direct binding assay could provide high throughput screening and reliable characterization of GLS1 inhibitors.

Microscale thermophoresis (MST)-based assay.56 MST method is another technology to assess the binding affinity of GLS1 and inhibitors. The method is usually employed to determine the binding affinity between protein-protein interactions or protein-small molecule interactions through detecting the change of fluorescence during thermophoresis.108 Chen et al. using the protein labeled with the Monolith NT™ Protein Labeling Kit RED (Cat#L001) under the manufacturer’s instructions to test the binding ability of inhibitor and purified KGA.56 The method could ensure the precise assessment of GLS1-ligand binding and provide useful tips for further optimization.

3.2 Cellular Assay 3.2.1 Cell-based Intracellular GAC Activity Assay Compared with the determination of the GLS1 inhibition activity in vitro, direct evaluation of GLS1 inhibition activity in cells is harder to achieve due to the complexity of cells. Recently, the ability of each inhibitor to affect intracellular GAC activity was performed by monitoring the level


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of ammonia in different cancer cells. Ammonia is the second product of GAC catalyzed hydrolysis of glutamine to glutamate.48 Drug-sensitive triple-negative breast cancer cell lines and the highly drug-resistant MDA-MB-453 cell lines were employed as model cell lines. When these cells were treated with inhibitors for a certain time, the amount of ammonia was determined with the Ammonia Assay Kit from Megazyme (Bray, Ireland). Effective GLS1 inhibitors would reduce the production of ammonia in both two cell lines, while the triple-negative breast cancer cells would be more sensitive to GLS1 inhibitors than drug-resistant (HER2-poisitve) MDA-MB-453 cells. 3.2.2 Inhibition of Cell Proliferation The anti-cancer cell growth and proliferation assays in vitro are so critical for the early effort of medicinal chemistry regardless of the drug-targets. With respect to glutaminase inhibitors, the selection of appropriate carcinoma cell lines is significant for stable and reliable cellular effects. Obviously, cancer cells that exhibit high expression of glutaminase or glutamine-addition are preferred for the anti-cancer cell growth and proliferation assays. It has been demonstrated that glutamine metabolism can be increased by Myc-induced the expression of glutaminase directly or suppression of miR23a and miR23b. Thus, P493 human B lymphoma cells, in which Myc is overexpressed in the absence of tetracycline, are used to assess antitumor effects of glutaminase inhibitors in vitro.54 TNBC primary tumors are reported to have high levels of glutaminase and low levels of glutamine synthetase. These protein expression levels suggest that the growth and proliferation of TNBC cells is highly dependent on glutamine. More importantly, the antiproliferative effects of CB-839 across a panel of 28 breast cancer cell lines (20 TNBC, 4 ER+/HER-, and 4 ER-/HER2+) has been evaluated and the result was that CB-839 shows antitumor activity toward most of the



TNBC cell lines with IC50 at 2-300 nmol/L.25 Thus, these results provide strong evidence that TNBC cell lines are a large class of reliable cells that can be used for rapidly screening anticancer activity of GLS inhibitors in vitro, of which the most commonly used are MDA-MB-231 and HCC1806. In addition, cells used to evaluate carcinoma cell growth inhibition activity of glutaminase inhibitors in literatures also include pancreatic cancer AsPC-1.26 Human hepatoma HepG2 and lung carcinoma A549 cells are found to show high expression levels of GAB and KGA protein.33 KGA was also found to be highly expressed in SW199055 and erlotinib-resistant NSCLC cells HCC827-ER.56 Finally, the methods for counting live cells mainly include CellTiter Glo Luminescent Cell Viability Assay kit (Promega) and trypan blue dye exclusion in a hemocytometer. 3.3 In vivo Assays The key point for in vivo anti-tumor growth and proliferation experiments of GLS inhibitors are still focused on the selection of tumor xenograft models. Just as in vitro carcinoma cell growth inhibition assay, a tumor xenograft model in vivo with high expression of glutaminase or high-dependence on glutamine is valuable for GLS inhibitors, mainly including TNBC,25 P493 Human Lymphoma B cells39 and AsPC-1 xenograft.26 In addition, it has been demonstrated that renal clear cell carcinoma cells, KRAS and EGFR mutant NSCLC lines exhibited more sensitivity to glutaminase inhibitors CB-839.58 The method of administration and dosage of drugs should also be determined based on the physicochemical and pharmacokinetic properties of compounds. Taking CB-839 as an example, due to its high clearance rate in mice, researchers take use of twice daily administration in order to maintain continuous target coverage.25 More importantly, the concern that synergistically anti-tumor proliferation study targeting


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glutaminase combined with other signaling pathways in vivo is more and more increasing. For example, CB-839 was co-administered with paclitaxel and achieved a 100% tumor inhibition rate in the JIMT-1 xenograft model.25 CB-839 shows a significant single-agent and combines with pomalidomide to produce strong anti-tumor activity in the IMiD-resistant RPMI-8226 xenograft model.57 In the paper, representative anti-tumor proliferation assays in vivo of glutaminase inhibitors are listed below (Table 4). Table 4. The representative anti-tumor proliferation assays in vivo of GLS inhibitors Tumor Xenograft Type

Drugs

P493 Human Lymphoma B cells

BPTES

AsPC-1

BPTES

TNBC PDX (CTG-0052)

CB-839 CB-839

JIMT-1

Paclitaxel Combination CB-839

RPMI-8226

H2122 (KRASmut) H1650 (EGFRmut, Erlotinib resistant model)

Pomalidomide Combination CB-839 Selumetinib Combination CB-839 Erlotinib Combination CB-839

HCC827 Erlotinib

Administration 12.5 mg/kg, i.p. every other day 25 mg/kg, i.p. Once daily on 5 on/2 off 200 mg/kg Orally BID 200 mg/kg Orally BID 10 mg/kg every other day for five doses + 200 mg/kg Orally BID 1 mg/kg Orally QD + precise data not shown precise data not shown 200 mg/kg, Orally BID 5 mg/kg, Orally QD


Tumor Growth Inhibition (TGI) precise data not shown39 55%26

61%25 54%

73% 100%25 64% 46% 97%57 46% 49% 78%58 26% 66% 89%58 precise data not shown59


Combination CB-839 Caki-1 (RCC)

Everolimus Combination CB-839

Caki-1 (RCC)

Cabozantinib Combination CB-839

HCT116

Palbociclib Combination CB-839

MCF-7

Palbociclib Combination CB-839

DU145

+ 200 mg/kg Orally BID 1 mg/kg Orally QD + 200 mg/kg Orally BID 1 mg/kg Orally QD + 200 mg/kg Orally BID 100 mg/kg Orally QD + 200 mg/kg Orally BID 50 mg/kg Orally QD + 200 mg/kg Orally BID

Talazoparib

0.25 mg/kg Orally QD

Combination

+

Page 20 of 65

precise data not shown60





TNBC, triple negative breast cancer; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; PDX, patient derived xenograft; RCC, renal cell carcinoma; TGI, tumor growth inhibition; BID, twice a day; QD, once a day; i.p., intraperitoneal. 4. Modulators of GLS In this review, we will highlight key tool compounds, clinical candidates, and new preclinical inhibitors for which potentially useful GLS1-inhibitors structural data have been reported. 4.1 Glutamine Mimetics 6-Diazo-5-oxo-L-norleucine (DON), azaserine and acivicin (Figure 4A) isolated from Streptomyces bacteria as glutamine mimetic, were reported to irreversibly interact with catalytic serine of both glutaminase isozymes and exhibit potent function in blocking glutamine


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metabolism.62,63 The previous reported crystal structure (PDB code: 4O7D) of the catalytic domain of KGA (cKGA) in complex with DON disclosed that DON could covalently bind with the active site Ser286 and have interactions with residues including Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484 (Figure 4B).52 The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and leads to the formation of an enzyme-DON complex (Figure 4C).52

Figure 4. (A) The structures of glutamine mimetics; (B) The interactions of DON with the catalytic site of KGA (cKGA, PDB code: 4O7D). DON is shown as a red stick and cKGA residues are shown in cyan; (C) Proposed active site inhibition mechanism of KGA.52 In preclinical studies, all of the three agents showed robust inhibitory effect in tumor models of glutamine-dependence both in vitro culture and in vivo mouse xenograft models.64,65 There were



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also some studies demonstrating that DON exhibited promising results in several clinical trials.66-72 However, the glutamine mimetic had severe off-target effects, e.g., DON could alkylate other enzymes except for GLS, such as NAD synthase,73 CTP synthetase74 and FGAR aminotransferase75 due to the over-active diazo group. Therefore, the clinical developments of DON and its diazo analogues were greatly limited due to dose-limiting toxicity and narrower therapeutic window.52,76,77 Although DON exhibited some off-target and side-effects as discussed above, the study on it has never been terminated. In 2016, Barbara S. Slusher and coworkers demonstrated that DON indeed had robust antagonist activity in glutamine metabolism.78 More importantly, it showed potent antitumor efficacy in a murine model of glioblastoma. In order to minimize the toxicity of DON in periphery, researchers adopted the most common prodrug strategy in medicinal chemistry leading to the obviously improvement in the blood-brain barrier permeability of designed DON derivatives and decrease in systemic exposure (Figure 5). Initially, compounds Rais-2a and Rais-2b (compounds in the paper are named by first author for academic publications, or patent assignee for patents, and then by the compound code within the relevant publication), just masking DON’s carboxylic acid with simple alkyl ester, were instable in chemical structure. Next, prodrug molecule Rais-5C (Figure 5) was synthesized by masking both amine and carboxyl groups of DON

with

prodrug

moieties

(methyl-pivaloyl-oxy-methyl

(POM)-DON-isopropyl-ester).

Although Rais-5C showed instability and was rapidly metabolized in mouse plasma, it was found that Rais-5C could be excellent stable in the plasma of human and monkey. Studies indicated that Rais-5C administration increased its brain delivery (1.43 nmol/g DON) compared to DON (0.33 nmol/g DON) in the cerebrospinal fluid (CSF) at 30 min postdose and decreased plasma exposure


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(AUC0-t = 5.71 nmol/h/mL) compared to DON (AUC0-t = 42.7 nmol/h/mL). In other words, prodrug molecule Rais-5C achieved a nearly 10-fold enhancement CSF to plasma ratio compared to DON in pigtail macaques.78

Figure 5. Chemical structures of glutamine analogues and the strategy of DON prodrugs. HIV-associated neurocognitive disorders (HAND) is associated with aberrant excitatory neurotransmission which is mainly related to the overexpression of glutamate. Based on the physiological mechanism, researchers proposed to attenuate glutamate production by DON so as to treat HAND. Firstly, researchers confirmed that DON indeed had significant effect in reducing cognitive decline in chimeric EcoHIV-infected mice, a model of HAND.79 However, the problem is still in DON itself with the peripheral toxicity. Similar to the content described above, researchers also adopted the strategy of prodrugs in order to achieve high blood-brain barrier penetration and low plasma exposure. Given some other successful prodrugs,80-83 modifications of the N-(pivaloyloxy)methoxy-carbonyl pro-moiety of Rais-5C with additional steric bulk on the methylene bridge were conducted. Compound Nedelcovych-13d (Figure 5) introducing phenyl

group exhibited the most desired activity. In addition to increasing steric hindrance and metabolic stability, lipophilicity of Nedelcovych-13d was also substantially increased compared with DON where the values of calculated partition coefficients (cLogP) of Nedelcovych-13d and DON were 2.75 and -2.5, respectively. Finally, to researchers’ most excitement, when dosed systemically in swine, Nedelcovych-13d provided a 15-fold enhanced CSF to plasma ratio and a 9-fold enhanced



brain to plasma ratio relative to DON. In sum, the two efforts described above open innovation directions for the development and application of DON on one hand. On the other hand the prodrug approaches provide new options for the treatment of central nervous system diseases associated with aberrant glutamine metabolism. 4.2 BPTES and Its Derivatives Based on screening of a library of chemical compounds, Robinson et al. discovered a potent and specific KGA inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide] with completely novel scaffold in 2007.24,84 The Ki value of BPTES in inhibiting KGA is highly potent with IC50 at 3 µM which is more potent than previously reported KGA inhibitors.76,85 In addition, the specific inhibition experiments indicated that BPTES treatment in 10 µM lead to 80% inhibition of KGA activity. Comparing the chemical structure with DON, BPTES does not possess any reactive chemical groups which might cause toxicity by irreversibly forming covalent adducts with the enzyme. Through further analysis the structure of BPTES, we find that it is a highly long, symmetrical, flexible and bears no similar group to substrate glutamine. This suggested that the interactions of BPTES with other glutamine-related enzymes, transporters, or receptors could be minimized so as to avoid off-target effects. Indeed, preliminary toxicity studies of BPTES in mice showed no histopathology in liver, heart, lung, skeletal muscle, kidney, and brain.86 Further, BPTES did not show any significant effects on body weight, blood chemistries, and hematology measurements. The X-ray crystallographic complex structures of mouse or human GLS1 with BPTES have been determined (PDB ID: 3UO9, 3VP1, 3VOZ, 5UQE and 4JKT). The X-ray crystal structures of the GLS1-BPTES complex show that BPTES occupies the allosteric


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binding site and effectively traps GLS1 as an inactive tetramer. The firstly reported co-crystal structure (PDB: 3UO9)47 of GAC with BPTES is shown in Figure 6. BPTES has two exactly equivalent parts including a thiadiazole, amide, and a phenyl group, which could equally interact with each monomer. The thiadiazole group and the aliphatic linker occupies well in the allosteric pocket and forms hydrogen bonds with Lys320, Leu321 and Leu323. When superimpose the BPTES-KGA complex structure with apo KGA, a major conformational change at the Glu312 to Pro329 loop was observed. The BPTES-induced conformational change was supposed to stabilize an open and inactive conformation of catalytic site (Figure 6C). The discovery of these co-crystal complex structures opens the way for the subsequent design and modification of BPTES analogues. However, studies on BPTES have been terminated in the preclinical phase due to poor metabolic stability and low solubility of BPTES.

Figure 6. (A) A close-up view of the interactions of BPTES in the GLS allosteric inhibitor binding pocket. (B) The chemical structure of BPTES. (C) Conformational changes on GLS induced by binding of the BPTES. (BPTES-KGA PDB: 3UO9; Apo GLS PDB: 3VOY).



In order to discover more potent GLS inhibitors with improved drug-like properties, Takashi Tsukamoto group designed a series of BPTES derivatives (Figure 7) and obtained the comprehensive SAR.54 The authors first attempted to identify the pharmacophore required for GLS1 binding, different truncated analogs of BPTES were synthesized and evaluated for their activity to inhibit GLS1. Compound Shukla-5, which was synthesized by removing the two phenylacetyl groups of BPTES scaffold, showed no potency in inhibiting GLS1. However, Shukla-6 (GLS-IC50: 2.7 µM) recovered the potency against GLS1 when introducing one of the phenylacetyl. The result demonstrated that one phenyl ring form BPTES was able to be removed without sacrificing potency. Such molecules maintain much of the BPTES scaffold with one hydrophobic end and one charged end. The two asymmetric molecules (Shukla-5 and Shukla-6) have not been determined by crystallographic studies, but have been supposed to bind the same location as BPTES. Next, they tried to explore the SAR of middle linker. According the inhibitory activity of compound Shukla-7 (GLS-IC50: 61 µM) and Shukla-11b (GLS-IC50: 1.9 µM), the length and type of the middle linkers were proved to play an important role in inhibiting GLS1. Moreover, different substitution groups were introduced into the phenylacetyl group of Shukla-11b. The results showed that the terminal aromatic ring was not necessary to maintain GLS inhibitory activity, which was consistent with Shukla-6. Researchers speculated that there was no specific interaction between the terminal substituent and the allosteric binding site. The fact that Shukla-20 and Shukla-27 synthesized by replacing the one of thiadiazole ring of Shukla-11b with 1,3-thiazole ring completely lost activity highlighted the crucial role played by N4 of the thiadiazole. In order to further investigate the effect of the thiadiazole ring on the activity, Shukla-29a was obtained by replacing one of the thiadiazole rings with a variety of amide groups.


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The results showed that the terminal thiadiazole can be replaced by other groups while maintaining the inhibitory activity of GLS. Finally, the aqueous solubility of Shukla-6, Shukla-11b and Shukla-29a (the values being 13, 3.4, 683 µg/mL, respectively) were indeed increased compared to BPTES (the value being 0.144 µg/mL). In summary, through Takashi Tsukamoto group early work, the aqueous solubility of the truncated analogues of BPTES was successfully improved, but none significant improvement in glutaminase inhibitory potency was achieved.54

Figure 7. Representative chemical structures of truncated BPTES analogs. In 2016, starting from the reported crystal structure of GLS1 in complex with BPTES, Takashi Tsukamoto group used 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)-butane as a core skeleton to explore the SAR of terminal phenylacetyl groups in an attempt to obtain additional interactions with the GLS allosteric binding site (Figure 8).87 The introduction of large steric hindered groups or positively charged groups into one of the phenylacetyl groups resulted in a loss or slight decrease of inhibitory activity against GLS1. Compounds Zimmermann-2h, 2i, and 2m containing hydrogen bond acceptor exhibited higher potency in GLS inhibitory with IC50 values



below 100 nM. The IC50 of Zimmermann-2j (GLS-IC50: 0.54 µM), synthesized by replacing phenolic group with acetoxy, was nearly 8-fold weaker than that of Zimmermann-2m (GLS-IC50: 0.07 µM). Based on the results, researchers speculated that the phenolic group of Zimmermann-2m might play an important role in interacting with GLS acting as both a hydrogen bond acceptor and a donor. Through the analysis of co-crystal complex structure of GLS1 with BPTES (PDB: 3VOZ)46, it is supposed that the phenolic group of Zimmermann-2m might bind with the guanidinium group of Arg317 and the carboxylate group of Glu325 that both located in the nearby of the phenolic group. In addition, the phenolic group also contributed to the improvement of the aqueous solubility of Zimmermann-2m (17 µg/mL) as compared to BPTES (0.14 µg/mL). Finally, researchers modified the phenylacetyl groups on both sides. The results showed that the both phenylacetyl groups can be modified without sacrificing the GLS inhibitor. More importantly, the fact that compound Zimmermann-2q (IC50 being 0.12 µM) with two phenols was less potent than Zimmermann-2m (IC50 being 0.07 µM) containing one phenol demonstrated that the substituents on both sides may have little synergistic effects on bounding to GLS.87


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Figure 8. Representative structures of compounds (Zimmermann-2h~2q) with 1,4-di(5-amino1,3,4-thiadiazol-2-yl)-butane scaffold. These compounds focused on exploring the SAR of terminal phenylacetyl groups. Further studies have demonstrated that the lipophilic connecting chains (diethylthio in BPTES) were the main cause of poor hydrophilicity and drug-like properties for these compounds.50 In order to improve the physicochemical properties, the flexible connecting chains were replaced by appropriate size ring systems. Bioactivity evaluations against GAC and MDA-MB-231 cell indicated that small to medium size heteroatom substituted rings are beneficial to activity, but too large rings are detrimental (Figure 9). Trans-CBTBP was synthesized by the replacement of aliphatic flexible linker on BPTES with 1,3-disubstituted cyclohexyl.49 Compared to BPTES, trans-CBTBP displays a smaller number of rotatable bonds (NRB, 8 in trans-CBTBP vs. 12 in BPTES). The reduction of NRBs in trans-CBTBP would improve the probability of good absorption. In addition, due to greater cell permeability, the IC50 for glutaminase inhibition for trans-CBTBP (IC50 = 0.1 µM) is only a moderate improvement over that for BPTES (IC50 = 0.16 µM). Among two trans-CBTBP enantiomers: 1S, 3S and 1R, 3R, only 1S, 3S-CBTBP was found to crystallize with cKGA (PDB code: 5JYP).49 This indicated that the 1S, 3S-CBTBP stereoisomer was preferred for cKGA over the 1R, 3R form. Compared to BPTES, 1S, 3S-CBTBP shared identical hydrogen bonding interactions with cKGA. However, the cyclohexane linker from 1S, 3S-CBTBP formed different hydrophobic interactions with the side chains of Tyr394, Phe322 and Leu321 from both the neighboring cKGA monomers compared to the aliphatic linker of BPTES.49 The activity against GAC of McDermott-7c (IC50 = 29 nM) containing 4-piperidinyl is more 10-fold than BPTES (IC50 = 371 nM).50 The physicochemical



properties, e.g., number of rotatable bonds (NRB)109, ligand efficiency (LE)110, lipophilic efficiency (LiPE)111, calculated octanol-water partition coefficient (ClogP), of the series analogues have indeed been improved compared to BPTES.

Figure 9. Representative compounds with appropriate size rings replacing flexible connecting linker. The human liver microsome (HLM) stability assay demonstrated that McDermott-7c exhibited more microsomal stability compared to BPTES. Finally, X-ray complex structures of


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GAC with constrained derivatives (McDermott-7d, McDermott-7e and McDermott-14d) confirmed that these compounds bind in the similar allosteric pocket just as BPTES (PDB ID: 5FI2, 5FI6 and 5I94).50 The ring linkers of these compounds lie in the same location of BPTES flexible connector chain. The thiadiazole groups can also occupy the same pocket of BPTES thiadiazoles in the 3UO9 X-ray structure (Figure 10). Overlaying the crystal structures and the variable orientation of the phenylacetic acid moieties suggested that the allosteric pocket of GLS1 is rigid, only the flexibility comes from the various inhibitors to better dock with the allosteric site to enhance the interactions. In view of the effectiveness of this strategy, many pharmaceutical companies also followed similar efforts. The specific contents are not reviewed here and can be seen in references.88-91

Figure 10. Overlay of binding modes of BPTES (pink, PDB: 3UO9), McDermott-7d (yellow, PDB: 5FI2), McDermott-7e (orange, PDB: 5FI6) and McDermott-14d (green, PDB: 5I94) with GAC. 4.3 BPTES Derivative in Clinical (CB-839) More recently, Gross et al. reported the development of CB-839, which was discovered from



several hundred BPTES derivatives.25 Currently, CB-839 is the only one GLS inhibitor that undergoing several different clinical studies.92,93 CB-839 is similar to BPTES in structure, just replacing one thiadiazole ring by pyridazine and replacing the two terminal phenyl rings by pyridine and trifluoromethoxy substituted phenyl ring. To explore the structural basis for the inhibitory efficacy, the complex structures of CB-839 with KGA and GAC have been solved (PDB ID: 5JYO and 5HL1).49 The conformations of CB-839 in KGA and GAC complex crystal structures are not exactly the same. According to both of the crystal structures, CB-839 was found to interact with the same allosteric pocket of GLS1 as reported to BPTES (Figure 11). The KGA-CB-839 (PDB ID: 5JYO)49 complex crystal structure shows that CB-839 could form hydrogen bonding contacts with the protein backbone amide groups of the Phe322 and Leu323. The pyridazinyl and acetyl groups of the inhibitor makes hydrogen bonds with Tyr394, Lys320 and Asn324. In addition, the thiadiazol group of CB-839 is also involved in a water-mediated interaction with Asp327. As for the terminal moieties of the trifluoromethoxy group, two different crystal structures CB-839-KGA and CB-839-GAC show inconsistent result (Figure 11). In the KGA-CB-839 complex crystal structure (PDB ID: 5JYO, Figure 11A)49, the trifluoromethoxy group is not engaged in any hydrogen bonding with KGA, while in the GAC-CB-839 complex (PDB ID: 5HL1, Figure 11B)49, the inhibitor-fluorine atoms can form hydrogen binding with Lys320. In addition, the trifluoromethoxy phenyl group is supposed to improve the solubility of CB-839.


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Figure 11. Co-crystal structures of GLS1 in complex with CB-839 bound to the allosteric pocket. (A) Detailed binding mode of CB-839-KGA (PDB ID: 5JYO); (B) Detailed binding mode of CB-839-GAC (PDB ID: 5HL1); Carbon atoms of CB-839 are shown in yellow stick. The residues involved in allosteric site were highlighted in rose red. Hydrogen bonds are represented by black dashed lines and H…π binding is represented by red dashed lines. (C) The structures of BPTES and CB-839. The biological experiment data disclosed that CB-839 is a potent, selective and orally bioavailable inhibitor of both KGA and GAC (the IC50 = 20 nM). In addition, CB-839 exhibited time-dependent inhibitory activity against GAC.25 This unique property did not appear in BPTES, and some researchers speculated that this may have a certain relationship with the pyridazine ring of CB-839. The in vitro antitumor activity toward triple-negative breast cancer (TNBC) cell lines HCC-1806 and MDA-MB-231 showed that TNBC were sensitive to CB-839.25 It should be noted



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that the sensitivity of TNBC cells toward CB-839 was correlated with two potential biomarkers, one of the marker is the overexpression of GAC but not KGA, the other is the high baseline ratio of glutamate to glutamine. Thus, these two markers that could be used to enrich for corresponding patients in clinical trials. Further in vivo antitumor experiment showed that CB-839 has potent antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2+ cell line model, JIMT-1, both showed antitumor activity as a single agent and in combination with paclitaxel.25 Moreover, the oral bioavailability and drug-like properties of CB-839 is excellent compared to BPTES. CB-839 is now in clinical trials for several different indications, both alone and as part of drug cocktails in multiple solid and liquid tumors.94 The reported clinical trials have been summarized in Table 5. Table 5. All reported Clinical Trials for GLS inhibitor CB-839a Clinical Trials. gov Identifier

NCT02071927

Status and Phase Completed Phase 1

Study Title

Study of the Glutaminase Inhibitor CB-839 in Leukemia

Conditions ·Acute Myeloid Leukemia (AML)

·Drug: CB-839

·Acute Lymphocytic

·Drug: CB-Aza

Leukemia (ALL)

A Comparative,

NCT02944435

Completed Phase 1

·Drug: CB-839

Pharmacokinetic Study of CB-839 Capsule and Tablet

·Healthy Volunteers

Formulations in Healthy

·Non-Hodgkin's

NCT02071888

Completed Phase 1

Inhibitor CB-839 in Hematological Tumors

Capsules ·Drug: CB-839 Tablets

Adults

Study of the Glutaminase

Interventions

·Drug: CB-839

Lymphoma (NHL)

·Drug: CB-839 and

·Multiple Myeloma

low dose

Waldenstrom's

dexamethasone

Macroglobulinemia

·Drug: CB-839,

(WM)

pomalidomide and

·Diffuse Large B-cell

low dose

Lymphoma (DLBCL),

dexamethasone

·Other B-cell NHL Subtypes, Including


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WM T-cell NHL

Recruiting NCT03047993

Phase 1 Phase 2

CB-839 + Azacitidine for Treatment of Myelodysplastic Syndrome (MDS) CB-839 + Capecitabine in

NCT02861300

Recruiting

Solid Tumors and

Phase 1

Fluoropyrimidine Resistant

Phase 2

PIK3CA Mutant Colorectal Cancer

NCT02771626

·Myelodysplastic Syndrome ·Other Diseases of Blood and

·Colorectal Cancer ·Colon Cancer

·Drug: CB-839

·Rectal Cancer

·Drug: Capecitabine

·Solid Tumor ·Clear Cell Renal Cell

Recruiting

Combination With

Carcinoma

Phase 1

Nivolumab in Patients With

·Melanoma

Phase 2

Melanoma, ccRCC and

·Non-small Cell Lung

NCT03163667

Recruiting Phase 2

With Everolimus in Patients

·Clear Cell Renal Cell

everolimus

Carcinoma

·Drug: Placebo plus everolimus

·Solid Tumors ·Triple-Negative Breast Recruiting Phase 1

Study of the Glutaminase

Cancer

Inhibitor CB-839 in Solid

·Non Small Cell Lung

Tumors

·Drug: Nivolumab

·Drug: CB-839 plus

With RCC (ENTRATA)

NCT02071862

·Drug: CB-839

Cancer

ENTRATA: CB-839 With Everolimus vs. Placebo

·Drug: Azacitidine

Blood-forming Organs

Study CB-839 in

NSCLC

·Drug: CB-839

Cancer ·Renal Cell Carcinoma Mesothelioma

·Drug: CB-839 ·Drug: Pac-CB ·Drug: CBE ·Drug: CB-Erl ·Drug: CBD ·Drug: CB-Cabo ·Drug: Glutaminase Inhibitor CB-839 ·Biological:

Novel PET/CT Imaging Biomarkers of CB-839 in Recruiting NCT03263429

Phase 1 Phase 2

Combination With Panitumumab and Irinotecan in Patients With Metastatic and Refractory RAS Wildtype Colorectal Cancer

·Colorectal Cancer ·Metastatic Colorectal Cancer ·RAS Wild Type Colorectal Cancer ·Refractory Colorectal Cancer

Panitumumab ·Drug: Irinotecan Hydrochloride ·Other: Laboratory Biomarker Analysis ·Other: Pharmacological Study ·Device: Imaging with 18F-FSPG PET/CT scans

NCT03428217

Recruiting

CANTATA: CB-839 With

·Advanced Renal Cell

·Drug: CB-839

Phase 2

Cabozantinib vs. Placebo

Carcinoma

·Drug: Cabozantinib



With Cabozantinib in Patients With Metastatic

·Metastatic Renal Cell

Page 36 of 65

·Drug: Placebo

Carcinoma

Renal Cell Carcinoma (CANTATA)

Study of CB-839 in Combination w/ Paclitaxel NCT03057600

Recruiting

in Patients of African

Phase 2

Ancestry and Non-African Ancestry With Advanced

·Triple Negative Breast Cancer ·TNBC-Triple-Negative

·Drug: Pac-CB

Breast Cancer

TNBC ·Drug: Glutaminase

CB-839 With Radiation

NCT03528642

Not yet

Therapy and Temozolomide

recruiting

in Treating Participants

(New)

With IDH-Mutated Diffuse

Phase 1

Astrocytoma or Anaplastic Astrocytoma

·Anaplastic Astrocytoma, IDH-Mutant ·Diffuse Astrocytoma, IDH-Mutant ·IDH1 Gene Mutation ·IDH2 Gene Mutation

Inhibitor CB-839 Hydrochloride ·Other: Questionnaire Administration ·Radiation: Radiation Therapy ·Drug: Temozolomide

a

The data is from www.clinicaltrials.gov

4.4 Benzophenanthridinone Scaffold Derivatives A novel small molecule 968, a dibenzophenanthridine, was identified as GLS inhibitor by screening for small molecule inhibitors of the transforming capabilities of activated Rho GTPases in 2010.35 Through the docking study together with mutational analysis of GAC, 968 is an allosteric regulator of recombinant GAC and its binding site lies in a cavity where two GAC monomers form a dimer that is different from above reported BPTES derivatives (Figure 12). SAR study indicated that the 3-bromo-4-(dimethylamino)phenyl ring of 968, also named ‘hot-spot’ ring, was significant for potency and required a large, antiplanar group at the para position for robust inhibitory potency (e.g., 968 vs Katt-14 vs Katt-22 in Figure 12).95 The biological result that introducing isopropyl (Katt-22) or tert-butyl (Katt-23) group at the position 4 of the hot-spot ring restored the enzyme activity demonstrated bulkiness could compensate for the loss of the


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bromine atom. Because Katt-26 bearing nitrile group exhibited no activity, it could be concluded that in addition to steric bulk, substituted groups must be oriented toward the para-position of the hot-spot ring. Moreover, researchers indicated that it is most effective to hold this steric bulk anti-planar to the hot-spot ring just as shown in Figure 12. Replacement of the naphthyl group of 968 could not significantly affect inhibitory activity (Stalnecker-SU14 or Stalnecker-SU8).96 It had been demonstrated that 968 had no inhibitory effect against glutaminase protein which have already been activated. This shortcoming and hydrophobicity limit the further application and continued research of 968 in the anti-tumor field.95-97

Figure 12. The structures of 968 and its representative derivatives. (A) SAR of



benzophenanthridinone scaffold derivatives. (B) Crystal structure of the GAC tetramer in complex with BPTES (PDB ID: 3UO9), with the proposed 968-binding site indicated by the arrow pointing toward the C-terminal monomer-monomer interface. BPTES and 968 were indicated by red and gray sphere, respectively. 4.5 Thiazolidine-2,4-dione Derivatives In 2017, Shiow-Ju Lee and coworkers discovered a series of thiazolidine-2,4-dione derivatives which were completely distinct from reported GLS inhibitors DON, BPTES or CB839 in chemical structure exhibited preferentially inhibitory activity against GLS1 over GLS2.26 Initial hit compound Yeh-2 was identified by a high-throughput screening (HTS) against KGA. Bioactivity evaluation indicated that the IC50 of Yeh-2 against KGA and GAB were 3097 and > 100000 nM, respectively. Yeh-3 replacing the methyl group of Yeh-2 with the thiophene group achieved an increase in the enzymatic inhibitory activity against KGA (IC50 = 754 nM). Further, compounds Yeh-5 and Yeh-6 were obtained by medicinal chemistry optimization of hit compound Yeh-2 (Figure 13A). They exhibited improved activity against KGA with the IC50 being 102 and 50 nM, respectively. Biochemical profiling indicated that Yeh-5 and Yeh-6 can inhibit tumor cell growth, including triple negative breast cancer MDA-MB-231 (the IC50 being 42 and 28.7 µM, respectively) and pancreatic cancer AsPC-1 (the IC50 being 34.5 and 14.8 µM, respectively) in vitro. Moreover, using the GLS1 knocked out cells AsPC-1GLS-/- cells, the selectivity of Yeh-6 was examined. The result shows that Yeh-6 can decrease the glutamate level both in AsPC-1WT cells and AsPC-1GLS-/- cells. However, BPTES can only decrease the glutamate level in AsPC-1WT cells but not AsPC-1GLS-/- cells. It is concluded that unlike BPTES, the selectivity of Yeh-6 for GLS1 and GLS2 is relatively weak with the selectivity index just being ~2. In human pancreatic AsPC-1


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xenograft antitumor assay, compound Yeh-5 and Yeh-6 exhibited about 50-60% tumor growth inhibition at a dose of 25 mg/kg or 50 mg/kg. Through molecular modeling and site-directed mutagenesis studies, researchers predicted that compound Yeh-5 may bind to the substrate binding site of KGA and interacts with R387 through hydrophobic bonding interactions (Figure 13B). This finding may open another potential binding site for novel allosteric inhibitors which bind GLS1 at the substrate binding pocket. The author also demonstrated when compound Yeh-6 was administered in combination with BPTES, a synergistic effect was also observed. In summary, this series of thiazolidine-2,4-dione compounds breaks through the structural framework of BPTES analogs described above and provides a novel backbone for the development of GLS inhibitors. However, its cell activity and enzyme activity remain moderate. Further efforts focus on structural modification and optimization of this scaffold are still needed.

Figure 13. (A) Representative glutaminase inhibitors of thiazolidine-2,4-dione scaffold. (B)



Overview of the proposed association of compound Yeh-5 in a GLS1 tetramer. 4.6 Other GLS1 Inhibitors from Natural Products Physapubescin and Brachyantheraoside A8 (Figure 14), two novel kidney-type glutaminase (KGA) inhibitors, were identified from natural products by the structure-based virtual screening.55,56 The enzyme inhibition experiments indicated that the IC50 values of physapubescin56 and brachyantheraoside A855 were 9.89 µM and 6.10 µM, respectively, with a closely similar effect compared with BPTES (IC50 = 8.60 µM). The cell-based assays revealed that the IC50 value of physapubescin against HCC827-ER and HT1080 cells were 2.73 µM and 6.99 µM, respectively and the IC50 value of against HCC1806 cell was 24.94 µM. In addition, a wound healing assay indicated that brachyantheraoside A8 treatment (30 µM) exerted robust effects in inhibiting HCC1806 cell migration ability. Finally, researchers confirmed that the anti-tumor activity of brachyantheraoside A8 is achieved by inducing cancer cell apoptosis through the modulating Bax/Bcl-2 ratio in a dose-dependent manner. Further evaluations of the two natural products in vivo need to be carried out. The discovery of these two KGA inhibitors from natural products may bring some new ideas for extending the structural diversity of GLS inhibitors.

Figure 14. The structures of two KGA inhibitors isolated from natural products.


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4.7 Natural Alkyl Benzoquinones as GLS2 Inhibitors The efforts in GLS2 inhibitors are limited in that the role of GLS2 as a tumor suppressor is still controversy. In this review, we would also take a brief introduction of GLS2 inhibitors. A series of natural products alkyl benzoquinones isolated from Ardisia virens exhibited potent and selective potency against recombinant human GLS2 enzyme.33 SAR study (Figure 15) indicated that introducing the keto (hydroxyl) groups at position 1 and 4 on the benzoquinone scaffold and the acetate group at position 2’ contribute to the inhibitory activity for KGA and GLS2. Compound Lee-AV1 (also namely as ardisianone) exhibited almost 10-fold selectivity against GLS2 (IC50 being 0.28 µM) and KGA (IC50 being 2.1 µM). Moreover, through homologous modeling and docking studies, researchers proposed that the binding pocket of Lee-AV1 with GLS2 distinct from the reported sites for BPTES, 968 or DON on GLS was located at the C-terminal end of GLS2 monomer (Figure 15) and the selectivity of Lee-AV1 for GLS2 over KGA was mainly related to GLS2 residues Q452 and K453. Finally, to further explore the mechanism how Lee-AV1 inhibit cancer cell growth, researchers examined some signaling factors associated with autophagy and apoptosis.98,99 Lee-AV1 treatment can induce autophagy by activating AMPK ULK1 axis signaling and inhibiting mTORC1, but no apoptosis. Thus, the autophagy was responsible for the inhibition of malignant cells growth.



Figure 15. The structure of alkyl benzoquinones and the mechanism that AV-1 inhibits glutaminase-2. 5. Future Directions and Conclusions Due to the unique characteristics of rapid proliferation and differentiation, cancer cells inevitably reprogram metabolic mechanisms to meet the energy needs for cell growth and maintain the balance of redox homeostasis. Glutamine, the most abundant amino acid in plasma, is a versatile nutrient required for the survival and growth of a potentially large subset of tumors. Moreover, the importance of glutamine as a nutrient in cancer derives from its abilities to donate its nitrogen and carbon into an array of growth-promoting pathways. It has been shown that cancer cells exhibiting resistant in chemotherapy is the upregulation of compensatory pathways when metabolic stress is induced by chemotherapy. Given this situation, some studies have demonstrated the combination of glutamine metabolism modulators and chemotherapy drugs may be a promising strategy to suppress the development of resistance to conventional chemotherapy for cancer.8,100,101 In addition to circumvent the resistance of chemotherapy, another important role of glutamine metabolism in cancer treatment is that it can contribute to sensitivity in radiotherapy.102 In various small molecule modulators targeting glutamine metabolism pathways, GLS1 inhibitors are the most widely studied, fastest developed and largest amount.3,20 Whether in preclinical or current clinical studies, they have demonstrated a promising role in the treatment of tumors, especially tumors that are highly dependent on glutamine.25 GLS1 possess a conserved substrate binding site46 and such structure poses a key challenge in designing selective GLS1 inhibitors. Thus, the discovery of inhibitors targeting allosteric binding site could be new strategy.


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The recent great progress in GLS1 crystallography has solved 16 GLS1 (including GAC and KGA) in complex with their small-molecule allosteric inhibitors.23 These solved receptor-inhibitor structural complexes pave the way for structure-based drug design or virtual screening to identify novel allosteric leads and to improve the binding affinity to existing allosteric modulators. Among the reported GLS1 allosteric inhibitors, CB-83925 is the first-in-class GLS1 inhibitor in glutamine metabolism, displays on-target cellular activity as indicated by its ability to suppress key glutamate-derived metabolic intermediates that support macromolecule synthesis, ATP production, and cellular redox balance. However, by now, nearly all preclinical studies or clinical studies have focused on the scaffold of BPTES and its derivatives.24,49,50,54,87 Several scaffolds have never been used after their initial report, probably because of specificity and/or poor drug-like properties in follow-up studies. Hence, the discovery of novel chemotypes serving as GLS1 inhibitors is urgent needed. To support the quest for novel GLS1 inhibitors, possible strategies in the scope of medicinal chemistry would be employed, such as scaffold hopping, bioisosteric replacement, combination of high throughput and in silico screening, and so on for the design of GLS1 inhibitors. To accelerate future progress, the assays to monitor GLS1 modulation have also been reviewed. In the next period, further studies around novel scaffolds are anticipated to be discovered to obtain the deep insight into the mechanism of GLS1 activation. In the future, except for waiting the progress of CB-839 in clinical which may bring a new research direction for the treatment of cancer, the combination strategy should also get some attention. Due to tumor cells could bypass the need for glutamine through different oncogenic drivers, recent findings have proved that inhibition of GLS alone is insufficient to halt progression of some tumors. Thus, inhibiting multiple targets in glutamine metabolism pathway synergistically



may be a more effective strategy to employ the glutamine metabolism to cancer therapy. While researching GLS1 inhibitors, several other inhibitors focus on the different targets in glutamine metabolism have been well studied, e.g., the discovery of V-9302,103,104 the first pharmacological inhibitor of the glutamine transporter ASCT2, substantially supported that antagonizing glutamine metabolism at the transporter level represents a potentially viable approach in precision cancer medicine. Combination of this inhibitor with efficient GLS1 inhibitors could be a potential strategy for pairing patients harboring glutamine dependent tumors. The huge therapeutic potential of glutamine metabolism has led the drug discovery on it. The glutaminase enzymes are the key players in facilitating the use of glutamine as an energy source, especially for GLS1, which is now the research hot-spot in this field. By now, though several potent inhibitors have been developed, the drug-like properties need to be optimized. Furthermore, the GLS1 inhibition or the exhaustion of glutamine metabolism pathway have been identified, but the applicable patient still need to be clarified. It is of paramount important for glutamine-based imaging into clinical practice to differentiate tumors that take up glutamine from those that do not. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Z. Li); [email protected] (J. Bian). Address: China pharmaceutical University, Nanjing. Author Contributions Jinlei Bian and Xi Xu are responsible for writing the whole passage. Zhiyu Li, Jinlei Bian and Ying Meng are in charge of checking and revision. Ying Meng contributes much work to create figures of glutamine metabolism pathway.


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Notes The authors declare no competing financial interest. Biographies Xi Xu received his Master’s degree at China Pharmaceutical University in 2017. Currently, he is pursuing his Ph.D. in the major of Medicinal Chemistry under the supervision of Professor Zhiyu Li. His research topic is mainly focused on the design, synthesis and biological evaluation of potent and selective small molecule GLS1 inhibitors. Ying Meng got Bachelor degree from Shandong University of Traditional Chinese Medicine in 2017. She is currently a postgraduate student at the Department of Medicinal Chemistry (China Pharmaceutical University) under the supervision of Professor Zhiyu Li. Her research mainly focuses on the structural optimization and biological evaluation of GLS1 inhibitors. Lei Li got Bachelor degree from Huaqiao University in 2016. He went on to advanced study in medicinal chemistry, China Pharmaceutical University, in the research group of Professor Zhiyu Li. His research focuses on design and synthesis of androgen receptor antagonists for the treatment of prostate cancer. Pengfei Xu, a graduating student, will soon receive his master's degree at China Pharmaceutical University under the supervision of Professor Zhiyu Li. Meanwhile, he will also continue pursuing Ph.D. degree in our group. During his postgraduate stage, his research work mainly focused on the optimization of synthetic processes. Jubo Wang achieved his Master’s degree at China Pharmaceutical University in 2012. After working several years in pharmaceutical companies, he selected to study his Ph.D. in the major of Medicinal Chemistry under the supervision of Professor Zhiyu Li in 2016. His research topic is



mainly focused on the design, synthesis and biological evaluation of potent and selective CDK9 inhibitors. Zhiyu Li graduated from China Pharmaceutical University with his bachelor’s and master’s degrees in 1988. He received his Ph.D. in medicinal chemistry at the same institute in 2000. Currently, he is the director and a full professor of the Department of Medical Chemistry, School of Pharmacy at CPU. He has published more than 50 papers in peer-reviewed journals and owns approximately 30 granted patents. His major research interests focus on the discovery and development of targeted anticancer drugs. Jinlei Bian received his Bachelor’s degree from China Pharmaceutical University, where he also obtained his Ph.D. in Medicinal Chemistry under the supervision of Professor Qi-Dong You. He is currently a member of the Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University. He works as a medicinal chemist, and his main research topics focus on the following: (i) rational design of modulators targeting tumor metabolism and (ii) the chemical biology study of GLS1. ACKNOWLEDGMENTS We are thankful for the financial support of the National Natural Science Foundation of China (No. 81703347, 21672260 and 21372260), the financial support of the National Natural Science Foundation of Jiangsu Province of China (No. BK20170743 and BK20171393), the Fundamental Research Funds for the Central Universities (2632017PY13), the National Found for Fostering Talents of Basic Science (NFFTBS) of China (No. 201710316077).


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ABBREVIATIONS USED ALL, acute Lymphocytic leukemia; AML, acute myeloid leukemia; ASCT2, alanine-serinecysteine transporter 2; ASNS, asparagine synthetase; BID, twice a day; BLI, bio-layer interferometry; BPTES, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide; ClogP, calculated octanol-water partition coefficient; CSF, cerebrospinal fluid; DLBCL, diffuse large B-cell lymphoma; DON, 6-Diazo-5-oxo-L-norleucine; EDTA, ethylene diamine tetra acetic acid; EGFR, epidermal growth factor receptor; FRET, fluorescence resonance energy transfer; GAC, Glutaminase C; GAB, glutaminase B; GBM, glioblastoma; GDH, glutamate dehydrogenase; Gln, glutamine; Glu, glutamate; GLS, glutaminase; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvate transaminase; HAND, HIV-associated neurocognitive disorders; HLM, human liver microsome; H2O2, hydrogen peroxide; HRP, horseradish peroxidase; HTS, high-throughput screening; i.p., intraperitoneal; KGA/GLS1, kidney-type glutaminase; α-KG, α-ketoglutarate; LAT1, L-type amino acid transporter 1; LE, ligand efficiency; LiPE, lipophilic efficiency; LGA/GLS2, Liver-type glutaminase; MDS, myelodysplastic syndrome; MST, microscale thermophoresis; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NHL, non-hodgkin's lymphoma; NRB, number of rotatable bonds; NSCLC,

non-small

cell

lung

cancer;

PDX,

patient

derived

xenograft;

PIK3CA,

phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene; PSAT1, phosphoserine aminotransferase 1; QD, once a day; RCC, renal cell carcinoma; SLC1A5, solute carrier family 1 neutral amino acid transporter member 5; TCA, tricarboxylic acid; TGI, tumor growth inhibition; TNBC, triple negative breast cancer; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine.



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Overview of the Development of Glutaminase Inhibitors: Achievements

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