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. 2022 Dec 22;14(1):11–17. doi: 10.1021/acsmedchemlett.2c00302

Synthesis of Novel Kidney-Type Glutaminase Allosteric Inhibitors Targeting the Critical Lys-320 Residue

Jun Song 1, Chuqiao Pan 1, Jinxiu Li 1, Ruisong Bai 1, Ziying Zeng 1, Yunying Han 1, Zhao Chen 1, Wei Hou 1, Yong Li 1, Benfang Helen Ruan 1,*
PMCID: PMC9841584  PMID: 36655131

Abstract

graphic file with name ml2c00302_0006.jpg

Reversible allosteric inhibitors of kidney-type glutaminase (GLS1, KGA) showed incomplete inhibition of cancer cell proliferation and poor in vivo efficacy. Here, we investigate some irreversible inhibitors targeting the critical K320 residue responsible for GLS1 biological activity. The (trifluoromethoxy)phenylacetic acid motif was replaced by α,β-unsaturated carboxylic acids, and the resulting terminally substituted CB839 derivatives (e.g., GJ2 and GJ5) showed good stability in solid form at room temperature, and better liver microsome stability and in vivo pharmacokinetics than coumarin. Both compounds showed binding to the wild-type KGA, whose KD is 106-fold stronger than that of CB839, but only weak binding to the KGA K320A mutant and no inhibition of GDH proteins. Interestingly, GJ2 treatment significantly decreased the trypsin digestion of KGA, tumor cell clonal formation, and cancer cell growth rate. Taking these results together, targeting the critical K320 residue of GLS1 might be a new strategy to make a potent GLS1 allosteric inhibitor.

Keywords: GLS1, allosteric inhibitor, CB839, antitumor, covalent inhibitor with Lys residue

Glutamine metabolism is important for tumorigenesis,1 and the key enzyme kidney-type glutaminase (KGA/GAC) has long been considered as a promising target for cancer therapy.2−4 DON is a KGA active-site inhibitor, with covalent binding to the KGA target and potent antitumor activity in animal models.5,6 However, due to its off-target effect (e.g., liver-type glutaminase, LGA), DON has strong gastrointestinal toxic side effects, but DON’s stable prodrug (methyl-POM-DON-isopropyl-ester) showed reduced toxicity and good biological activity.7,8

KGA allosteric inhibitors such as BPTES9 and CB839, as shown in Figure S1, have attracted great attention due to their low toxic side effects.10,11CB839 has shown some efficacy in in vivo cancer models, such as the glutamine-dependent triple-negative breast cancer, hematological tumors, and other solid tumors.12 Recently, Soth et al.13 developed a new compound, IPN60090 (GLS, IC50 = 31 nM), that is currently in Clinical Phase I. IPN60090 showed high selectivity for GLS1 (IC50 = 31 nM) but no activity against GLS2. Bian Jinlei’s team recently reported14 a series of macrocyclic GLS1 inhibitors 13b created by linking the terminal heteroaromatic rings (Figure S1), which have significantly improved water solubility and improved pharmacokinetic (PK) properties. However, both IPN60090 and 13b, dosed orally at 200–250 mg/kg per day, could only achieve in vivo efficacy similar to that of CB839. The key problem resulting in poor in vivo efficacy is that the reversible inhibitors could not completely inhibit cancer cell proliferation.15 The observed drug-induced proliferation rate (DIP) for an overnight culture containing CB839 was still significant, which was more than 10% of the growth rate of the untreated cells.

Interestingly, hexylselen, a dual KGA/GDH inhibitor, was able to completely inhibit cancer cell proliferation and significantly decrease KGA protein levels in the treated cancer cells.16 Therefore, we are interested in investigating if modifying the reversible inhibitor CB839 into an irreversible inhibitor could further deplete the KGA enzyme and decrease the DIP rate. The commonly used chemical strategy for forming a covalent linkage is targeting the cysteine (Cys) residue, such as the KRAS inhibitor AMG 51017 and the BTK kinase inhibitor that covalently link the cysteine residue to the acrylamide group of the lead compound;18 both have successively entered clinical research. The advantages of covalent inhibitors are the high bonding efficiency, prolonged action time,19−21 and reduced drug resistance caused by target mutations.

We designed the irreversible inhibitor by targeting the critical KGA residue Lys-320, which is at the activation loop. The K320 loop was randomly positioned until an allosteric inhibitor (e.g., CB839) was bound. As shown by the co-crystal structure, two molecules of CB839 interact with cKGA at its tetramer interface;22 the nitrogen atoms in the thiadiazole ring and pyridazine ring of CB839 can form hydrogen bonds with the amide groups of Phe322 and Leu323, and the pyridazinyl and acetyl groups of the compound interact with the side chains of Try394, Lys320, and Asn324 of the cKGA protein backbone.5,23 Besides Lys320, other Lys residues at the KGA tetramer interface might be also important,24 and these lysine residues could also provide nice handles for an irreversible KGA allosteric inhibitor. Therefore, we designed and synthesized a series of GJ2 and GJ5 derivatives containing an acrylamide Michael acceptor. In comparison with CB839, both irreversible inhibitors selectively targeted the K320 residue, significantly reduced the rate of cancer cell proliferation, induced cellular reactive oxygen species (ROS), and effectively inhibited tumor cell clone formation. These new findings in these works provide important insight for future development of an effective KGA allosteric inhibitor.

Chemistry

The irreversible KGA inhibitors were synthesized as shown in Figure 1. The core structure of compound 5 was synthesized using the procedures reported previously,25 and the irreversible inhibitors were synthesized through peptide bond formation between a series of α,β-unsaturated carboxylic acids and 5. The peptide formation reaction was carried out by mixing amine, acid, and HBTU in DMF, and then treating with DIPEA, followed with heat treatment at 60 °C overnight. After TLC assay showed the completion of the reaction, the reaction was terminated by adding saturated brine to precipitate the solid product. The solid was collected by filtration, and the filtrate was extracted with ethyl acetate. The crude product was dissolved in DCM/MeOH and purified by column chromatography (DCM:MeOH = 50:1). A yellow solid of compound 6 was obtained in 31% yield. This peptide formation procedure was used for all compounds listed in Figure 2.

Figure 1.

Figure 1

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Synthetic scheme of the covalent KGA inhibitors. Reagents and conditioins: (a) POCl3, reflux; (b) EDCI, DMAP, DIPEA, RT; (c) PdCl2(PPh3)2, CuI, TEA, reflux; (d) Raney-Ni, H2; (e) HBTU, DIPEA, 60 °C.

Figure 2.

Figure 2

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Designed and synthesized covalent KGA inhibitors.

Among the structures, the (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid derivatives (GJ2 and GJ5) were stable, whereas the terminal unsubstituted acrylic acids GJ1, GJ3, GJ4, and GJ7 were not stable at room temperature. GJ6 was not stable enough to be purified to >95% purity. Therefore, GJ2 and GJ5 were further characterized in detail and used for mechanistic investigations exploring covalent bond formation with the Lys320 residue of the KGA enzyme.

Biology

The selectivity of these irreversible inhibitors was investigated by inhibition assays using both human KGA and the native bovine GDH enzymes. The compounds show good inhibition of KGA but no inhibition of GHD, as shown in Figure S2 and Table S1, indicating these irreversible inhibitors have good KGA target selectivity; compounds GJ2 and GJ5 showed especially strong KGA inhibition with IC50 values of 0.05 μM and 0.028 μM, respectively.

Further, the irreversible inhibitors GJ2 and GJ5 were shown to be able to selectively target the Lys320 residue of the wild-type KGA. As shown in Figure S3, the cloned and expressed KGA mutants (K320A and K320G) showed lower activity than the wild-type KGA, although the activity of mutant K320A was better than that of K320G, indicating that the chemical nature of the molecule covalently linked to the Lys320 could also affect the KGA enzyme activity. We selected the K320A mutant for further studies, because the mutant protein still has weak activity and the Ala residue could not form a covalent bond with GJ2 and GJ5. However, to achieve the same protein activity as the wild-type KGA protein, we used 10 times more mutant K320A protein in the assays and 30 times more GJ2 and GJ5 compounds than the K320A protein.

Interestingly, GJ2 and GJ5 showed potent inhibition of the wild-type KGA but poor inhibition of the mutant K320A enzymes. As shown in Figure 3A,B, in the presence of 10 μM GJ2 or GJ5, the activity of the wild-type KGA was completely inhibited, but the mutant KGA showed essentially the same enzyme activity in the presence or absence of the compounds; this indicates that the K320 residue is important for forming the covalent bonding, and its covalent bond formation with the compound completely inhibited the wild-type KGA enzyme activity. The mutant KGA-K320A enzyme does not have a K320 residue, and therefore cannot be inhibited by this covalent binding The critical role of the K320 residue in mediating the action of GJ2 or GJ5 on the enzyme was further confirmed by the biomolecular interaction assay (BLI technology).

Figure 3.

Figure 3

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Biochemical characterization. (A, B) Enzyme activity tests of the human KGA enzyme (wild-type or K320A mutant) in the presence or absence of either GJ2 or GJ5, showing that pre-incubation with the compounds inhibited the wild-type enzyme but not the K320A mutant. (C, D) Trypsin digestion of KGA in the presence or absence of GJ2, showing that GJ2 prevented the trypsin digestion and indicating that GJ2 might form a covalent linkage with the Lys residue of the wild-type KGA. (E–G) BLI assays showing the kinetic binding of CB839, GJ2, and GJ5 to the KGA protein. The overall curve fittings show that (E) CB839 (0–10 μM) has KD = 1.5 × 10–6 M, (F) GJ2 (0.1–10 μM) has KD ≤ 1 × 10–12 M, and (G) GJ5 (0.1–10 μM) has KD ≤ 1 × 10–12 M in PBS buffer containing 1% DMSO (blue, low concn, 1 μM; orange, middle concn, 3 μM; red, high concn, 10 μM).

The BLI assay was carried out using the method we developed previously.16,26 Excess amounts of compound GJ2 or GJ5 (3 μM) were tested with the KGA protein (40 nM) or the mutant KGA K320A protein (400 nM). The binding of the wild-type KGA or the mutant KGA K320A protein to the biotinylated CB839 was compared in the presence or absence of a series dilution of GJ2 or GJ5. The wild-type enzyme showed a huge binding difference in the presence of GJ2 or GJ5 (>300 nM), indicating that the compound completely blocked the enzyme from binding to the biotinylated CB839. Under the same conditions, the mutant KGA K320A protein showed nice dose response in the presence of GJ2 or GJ5 (0–3 μM), indicating that, in the absence of the K320 residue, both compounds become reversible inhibitors of the mutant KGA K320A protein (Figure S3).

In addition, CB839, GJ2, and GJ5 were used in direct KGA binding assays. As shown in Figure 3E, CB839 showed strong binding to the KGA protein in a dose-dependent manner with a significant off rate, whereas GJ2 or GJ5 also showed strong binding but with a very small off rate, resulting in a 106 times decrease in KD values, indicating covalent bond formation (Figure 3F,G). Taken together, both the activity assay and the BLI binding assay demonstrated that the covalent inhibitors GJ2 and GJ5 irreversibly bond to the KGA K320 residue, and GJ2 and GJ5 are stronger inhibitors against the reversible inhibitor CB839.

For further validation that GJ2 or GJ5 could cross-link the Lys residue at the KGA allosteric site, KGA was pretreated with GJ5, followed by trypsin digestion which cuts after the lysine residue. Interestingly, based on gel electrophoresis analysis, the digestion of the KGA was reduced by approximately 50% in the presence of GJ5 (Figure 3C,D), indicating the covalent linkage formation between KGA and the compound through the Lys residues.

The pharmacological improvement was tested in cell-based assays. Interestingly, the irreversible inhibitors GJ2 and GJ5 showed significantly improved maximal growth inhibition in comparison to CB839. As shown in Table S2, GJ5 treatment improved the IC50 values against HCT116 cells by 3–5-fold, and H22 cells by 30–100-fold. In addition, when the drug-induced proliferation rates (DIP rates) were measured, 10 μM GJ2 or GJ5 could essentially inhibit the growth (<10% growth) of the HCT116 cell, whereas 10 μM CB839 still showed significant growth (>10% growth) (Figure 4a–d and Table S3). At 3 μM concentrations, CB839 did not completely inhibit cell growth, with growth rates around 17%, indicating partial drug resistance, whereas under the same conditions, GJ2 and GJ5 showed significantly reduced growth rates of 6% relative to those of the untreated cells (Table S3). This result indicated that covalent inhibitors have greater advantages due to their prolonged pharmacodynamic effects.

Figure 4.

Figure 4

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Cell-based assays. (a–c) DIP rates of HCT116 cells in the presence or absence of (a) CB839, (b) GJ2, or (c) GJ5. Cells without compound treatment showed strong growth (DIP rate) and reached the maximum growth after the initial 2 h incubation, but in the presence of a compound (1, 3, or 10 μM) the DIP rates dropped significantly. (A–G) Compound treatment affected the colony formation of A549 cells. Colony formation was significantly reduced by GJ2 (A, B) or GJ5 (C, D) in comparison with the DMSO control group (E, F). (G) Statistical analysis of colony formation with or without drug treatment by an unpaired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (d) Comparative inhibitors of cell growth. DIP rates of GJ2 and GJ5 were reduced by more than 2-fold in comparison to that of CB839.

In addition, a colony formation assay was used to investigate the tumorigenic activity of compounds GJ2 and GJ5. Figure 4A–G shows that the drug treatment significantly reduced both the number and the size of the colonies formed in a dose-dependent manner. In the cellular ROS assay, GJ2 and GJ5 showed more ROS induction than CB839. As shown in Figure S5, compound GJ2 produced more ROS than CB839 at a concentration of 10 μM. This further indicated that effective inhibition of the glutaminolysis might result in blocking the mitochondrial TCA cycle and further increasing the ROS level.

The metabolic stability of compounds GJ2 and GJ5 was also investigated in liver microsomes and PK studies. HPLC analysis, after incubation of the compounds with mouse liver microsomes for 2 h, showed that the levels of GJ2, GJ5, and the control coumarin were dropped to 74%, 83%, and 61%, respectively, indicating that GJ2 and GJ5 have relatively good liver microsome stability (Figure 5a). In addition, a preliminary PK study showed that the calculated half-life of GJ5 was 2.6 times longer than that of coumarin (Figure 5b) and the corresponding clearance reduced by 2.6-fold, suggesting that GJ2 and GJ5 are relatively stable chemicals. Although the current PK studies were limited by the compound solubility, a good formulation remains to be developed to achieve good bioavailability.

Figure 5.

Figure 5

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Pharmacokinetic studies of (a) compound stability in a liver microsome assay and (b) in vivo PK parameters of GJ2 and GJ5 in mice.

In conclusion, due to their high selectivity between KGA and LGA or other glutamine binding enzymes, the allosteric inhibitors of KGA/GAC have attracted great attention in cancer therapeutics.13,14,25,29 Unfortunately, despite potent KGA inhibition, all reversible allosteric inhibitors showed limited efficacy in both cell proliferation assay and in vivo animal model.30 We aim to explore a novel approach by targeting the essential residue K320 located at the “activation” loop of the KGA. Based on crystal structure, we designed a series of CB839 derivatives (or selenium-containing derivatives) containing the α,β-unsaturated carboxylate motif for potentially a covalent bond formation with the K320 residue. Even though the terminal unsubstituted acrylic acids were not stable, the (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid derivatives GJ2 and GJ5 showed good stability. Interestingly, both compounds showed covalent linkage to the K320 residue of the wild-type KGA enzyme, resulting in at least 2–3 times stronger activity than the reversible inhibitor CB839 in various efficacy studies. This indicates that targeting the KGA K320 residue is achievable and the resulting irreversible inhibitor significantly improves drug efficacy.

Acknowledgments

The authors appreciate the financial support from Fuyang government innovation grant (H1160200772), and Natural Science Foundation of Zhejiang Province/General Project, China (number LY19H300002).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00302.

  • Experimental details; 1H and 13C NMR spectra of GJ2 and GJ5, 1H NMR spectrum of GH6, and HRMS spectra of GJ2, GJ5, and GJ6; Figures S1–S6 and Tables S1–S3 (PDF)

Author Present Address

‡ Allsio LLC, 1378 West Wenyi Road, Hangzhou 310014, PR China

Author Contributions

† J.S., C.P., and J.L. contributed equally. B.R. conceived the idea and performed data analysis. J.L., C.P., Y.H., Z.Z., and Z.C.: biology. J.S., R.B., W.H., and Y.L.: chemistry.

The authors declare no competing financial interest.

Supplementary Material

ml2c00302_si_001.pdf (951.2KB, pdf)

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Associated Data

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