ASCT2 (alanine-serine-cysteine transporter 2, encoded by SLC1A5) - primary transporter of glutamine in cancer cells. IC50 for glutamine uptake inhibition = 9.6 μM. [1]
SNAT2 (sodium-neutral amino acid transporter 2) - potential off-target. [2][3]
LAT1 (large neutral amino acid transporter 1) - potential off-target. [2][3]

V-9302 hydrochloride is 100 times more effective than γ-L-glutamyl-p-nitroanilide and inhibits ASCT2-mediated glutamine absorption in human cells in a concentration-dependent manner [1]. enhanced oxidative stress, reduced cancer cell growth and proliferation, and enhanced cell death are the outcomes of pharmacologically blocking ASCT2 with V-9302 hydrochloride [1].

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- V-9302 inhibited ASCT2-mediated glutamine uptake in HEK-293 cells in a concentration-dependent manner with an IC50 of 9.6 μM, showing 100-fold improvement in potency over GPNA (IC50 = 1,000 μM). [1]
- At concentrations up to 10-fold greater than its IC50 (100 μM), V-9302 preferentially inhibited glutamine transport and also inhibited uptake of another ASCT2 substrate, leucine. [1]
- DARTS (drug affinity responsive target stability) assay showed protection of ASCT2 from proteolytic degradation by thermolysin in a V-9302-concentration-dependent manner (50, 100, 200 μM), indicating a stable V-9302-ASCT2 interaction. The ASCT2 paralog, ASCT1, was not stabilized in the presence of V-9302. [1]
- In silico docking studies showed that V-9302 is compatible with the orthosteric amino acid-binding pocket of human ASCT2, localized within the transmembrane region. The conserved α-amino acid headgroup forms key interactions within the zwitterion recognition site. [1]
- V-9302 exposure (25 μM, 48 h) in HCC1806 cells resulted in decreased phosphorylated S6 (p-S6) levels and modestly decreased p-ERK levels, analogous to ASCT2 shRNA knockdown. [1]
- V-9302 exposure led to increased levels of oxidized glutathione (GSSG) at the expense of reduced glutathione (GSH), and a corresponding increase in intracellular reactive oxygen species (ROS) in HCC1806 and HT29 cells. [1]
- V-9302 exposure (10 or 25 μM, 48 h) led to elevated expression of LC3B (autophagy marker) and decreased the optical redox ratio ([FAD]/[NAD(P)H]) in HCC1806 cells. [1]
- In a panel of 29 human cancer cell lines (lung, breast, colorectal cancer), V-9302 (25 μM, 48 h) reduced ATP-dependent viability by at least 20% in more than half of the cell lines. EC50 values for four colorectal cancer cell lines ranged from approximately 9-15 μM. [1]
- In glutamine-dependent liver cancer cell lines (SNU398, HepG2), the combination of V-9302 and the GLS1 inhibitor CB-839 showed synergistic anti-proliferative effects, depleting glutathione and inducing lethal levels of reactive oxygen species (ROS) and DNA damage (γ-H2AX). NAC (ROS scavenger) rescued cell viability and proliferation. [2]
- In triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC1806, 4T1, E0771), V-9302 induced cell death in a dose-dependent manner. [3]
- V-9302 (10 μM) did not impact the viability of activated CD8+ T cells or CD4+ T cells, nor did it affect mTORC1 signaling (phospho-S6RP) in T cells. [3]
- V-9302 increased glutamine uptake in CD8+ T cells (compensatory upregulation of ATB⁰⁺/Slc6a14), while decreasing glutamine uptake in TNBC cells (E0771, HCC1806). [3]
- V-9302 increased glutathione levels, intracellular cysteine, GCLC expression, and decreased ROS in activated CD8+ T cells. [3]

In both the HCT-116 and HT29 xenograft models, V-9302 hydrochloride (75 mg/kg; i.p.; once daily for 21 days) inhibits the growth of tumors [1]. SNU398 and MHCC97H cells produced as tumor xenografts in BALB/c nude mice; cultivated for 20 or 15 days, respectively) in combination with CB-839 and V-9302 hydrochloride (30 mg/kg; i.p.). caused a significant growth inhibition in transplantation models; however, the antitumor effects of a single medication therapy were only moderate [2]. Tumor growth was significantly reduced when V-9302 hydrochloride (50 mg/kg; i.p.; once daily for 5 days) was administered [3].

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- Pharmacodynamic PET imaging: In athymic nude mice bearing HCC1806 xenografts, a single dose of V-9302 (75 mg/kg body weight, i.p., 4 h) reduced tumor uptake of [4-¹⁸F]fluoroglutamine by approximately 50%, reaching levels below background uptake in healthy muscle. Muscle uptake was unaffected, while liver uptake was modestly elevated (P = 0.05). [1]
- Chronic efficacy in HCT-116 xenografts: Athymic nude mice bearing HCT-116 (KRAS-G13D) xenografts were treated with V-9302 (75 mg/kg/day, i.p., daily for 21 days). V-9302 prevented tumor growth compared to vehicle, with significantly decreased p-S6 expression and elevated cleaved caspase-3 in tumor tissue. [1]
- Chronic efficacy in HT29 xenografts: Athymic nude mice bearing HT29 (BRAF-V600E) xenografts treated with V-9302 (75 mg/kg/day, i.p., daily for 21 days) showed prevented tumor growth, decreased p-S6, and elevated cleaved caspase-3. [1]
- Patient-derived xenograft (PDX) efficacy: In athymic nude mice bearing A-008 PDX (KRAS-G12V; p53-R248Q; PTEN-L140Y), V-9302 (75 mg/kg/day, i.p., daily for 31 days) led to reduced tumor volume compared to vehicle. [1]
- HCC1806 and COLO 205 xenografts: V-9302 treatment for 10 days arrested tumor growth, characterized by elevated LC3B, cleaved caspase-3, and decreased p-Akt (Ser473) and p-S6. In COLO 205 xenografts, decreased p-ERK and BrdU uptake, and elevated p-GSK-3β, p-PRAS40, p38, and p53 were observed. [1]
- In a spontaneous mouse TNBC model (C3(1)-TAg), mammary epithelial-specific GLS loss (genetic model) delayed tumor latency and improved T cell activation. Pharmacological treatment with V-9302 (50 mg/kg, i.p., daily for 5 days) in E0771 tumor-bearing C57BL/6 mice reduced tumor growth and increased intratumoral CD8+ T cell infiltration and effector function (GZMB, CD107a, IFN-γ). [3]
- In SNU398 and MHCC97H liver cancer xenografts (BALB/c nude mice), the combination of V-9302 (30 mg/kg, i.p., daily) and CB-839 (150 mg/kg, oral gavage, twice per day) for 20 or 15 days resulted in strong growth inhibition, decreased Ki67, and increased cleaved caspase-3 and γ-H2AX in tumor tissues. No body weight reduction was observed. [2]

- 3H-labeled amino acid uptake assay: Live-cell amino acid uptake assays were carried out in 96-well plates using HEK-293 cells. Cells were washed with assay buffer (137 mM NaCl, 5.1 mM KCl, 0.77 mM KH₂PO₄, 0.71 mM MgSO₄·7H₂O, 1.1 mM CaCl₂, 10 mM D-glucose, 10 mM HEPES). ³H amino acid (500 nM) was added concomitantly with V-9302 and incubated for 15 min at 37°C. For ASCT2-mediated ³H glutamine uptake assays, 5 mM BCH (system L inhibitor) was added and buffer adjusted to pH 6.0. For selectivity studies, no BCH was added and assay was conducted at pH 7.4. Cells were lysed with 1 M NaOH, scintillation fluid added, and counted on a scintillation counter. IC50 values were calculated using GraphPad Prism. [1]
- DARTS (Drug Affinity Responsive Target Stability) assay: T-REx-293 cells with tetracycline-inducible ASCT2 expression were used. Lysates were exposed to V-9302 at varying concentrations (50, 100, 200 μM) for 35-45 min at room temperature with shaking, then incubated with thermolysin (1:100 to 1:200 enzyme:substrate) for 30 min. ASCT2 was measured by immunoblotting. [1]
- In silico docking: A homology model of human ASCT2 was used as a target for ligand docking. 2D structures were generated in ChemDraw, converted to 3D structures, and docked using RosettaLigand. Fragment constraints were used to encourage placement of the amino acid analog main-chain atoms analogous to TFB-TBOA in hEAAT1. For comparative studies of ASCT2 and LAT1, homology models were generated using MOE. Ligand rotational conformer libraries were generated and binding sites identified. Each simulation consisted of 500 iterations scoring protein-ligand interface energies. [1]
- Metabolomics by LC-MS: Cells were treated with DMSO or CB-839 for 4 and 24 hr. Metabolites were extracted in methanol/acetonitrile/dH₂O (2:2:1), centrifuged, and supernatants analyzed on an Exactive mass spectrometer coupled to a Dionex Ultimate 3000. Separation used a SeQuant ZIC-pHILIC column with a linear gradient of acetonitrile and 20 mM (NH₄)₂CO₃, 0.1% NH₄OH. Metabolites were identified and quantified based on exact mass within 5 ppm and retention time concordance with standards. [2]
- Glutamine uptake assay in coculture: E0771 or HCC1806 cells were plated in 96-well plates. CD8+ T cells were added to tumor cells (1:1 ratio) in uptake buffer with V-9302 (10 μM), MeAIB (10 mM), BCH (500 μM), or αMT (100 μM). After 15 min incubation at 37°C, 1 μCi of L-[2,3,4-³H]-glutamine was added for 15 min. Cells were washed, lysed with 1N NaOH, and radioactivity measured by scintillation counter. [3]

- Cell viability assay (ATP-dependent): Cells were exposed to V-9302 (25 μM, 48 h) in 96-well plates. CellTiter-Glo reagent was added and plates read using a plate reader. [1]
- CellTiter-Blue viability assay: Cells were seeded in 96-well plates (2,000-5,000 cells per well). After 12 h, indicated drugs were added. Cell viability was measured after 72 h. Relative viability was normalized to control conditions. [2]
- Long-term colony formation assay: Cells were seeded onto 6-well plates at 2-10 × 10⁴ cells per well and cultured with indicated drugs for 10-14 days (medium changed twice a week). Cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet. [1][2]
- IncuCyte cell proliferation assay: Cells were seeded onto 96-well plates at 1,000-8,000 cells per well. After 12 h, drugs were added and cells imaged every 4 h. Phase-contrast images were analyzed to detect cell proliferation based on cell confluence. [1][2]
- IncuCyte apoptosis assay: Caspase-3/7 green apoptosis assay reagent was added to culture medium. Apoptosis was analyzed based on green fluorescent staining of apoptotic cells. [2]
- Glutathione and ROS measurement: Glutathione levels were measured using a commercial kit (Cayman Chemical). ROS were measured using CM-H₂DCFDA coupled with flow cytometry. [1]
- Flow cytometry for T cell activation: Tumors were harvested, dissociated in RPMI-1640 with 5% FBS, collagenase IA (1 mg/mL), and DNase I (0.25 mg/mL) for 30 min at 37°C. Single-cell suspensions were filtered, RBCs lysed. For intracellular cytokine detection, cells were stimulated with PMA (50 ng/mL), ionomycin (1 μg/mL), and GolgiPlug for 4 h at 37°C. Cells were stained with Ghost Dye for viability, Fc-blocked, then stained with antibodies against CD45, CD3e, CD4, CD8a, CD25, CD69, CD44, CD62L, CD127, CD107a. Intracellular staining for GZMB, IFN-γ, IL-4, IL-17A, and GCLC was performed using Cytofix/Cytoperm. FoxP3 staining was performed using FoxP3/Transcription Factor Staining Kit. Data acquired on BD Fortessa and analyzed with FlowJo. [3]
- Cytotoxicity assay (LDH release): E0771(OVA) or E0771 cells (1 × 10⁴ per well) were plated and cocultured with SIINFEKL-activated CD8+ OT-1 T cells (2 × 10⁵ per well; 5:1 ratio) with or without V-9302 (10 μM) for 48 h. Cytotoxicity was measured using LDH Cell-mediated Cytotoxicity Assay. [3]
- Quantitative real-time PCR: RNA was collected using RNeasy kit, cDNA generated using iScript cDNA synthesis kit. Samples amplified in triplicate using SYBR Green PCR Master Mix with primers for Slc1a5, Slc38a1, Slc38a2, Slc7a5, Slc3a2, Slc6a14, and GCLC. Quantitation performed using ΔΔCt method. [3]

Animal/Disease Models: 6-week old, female athymic nude mice (bearing HCT-116 (KRASG13D) or HT29 (BRAFV600E) cell-line)[1]
Doses: 75 mg/kg
Route of Administration: intraperitoneally (ip); daily fo 21 days
Experimental Results: Prevented tumor growth.

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- In vivo efficacy in xenograft models: Athymic nude mice (Hsd:Athymic nude-Foxn1nu) bearing HCT-116, HT29, HCC1806, or COLO 205 cell line xenografts were treated with V-9302 (75 mg/kg body weight per day, i.p., daily for 21 days, or 10 days). For PDX model A-008, treatment was for 31 days. V-9302 was reconstituted in PBS supplemented with 2% DMSO. [1]
- PET imaging study: Mice bearing HCC1806 xenografts were administered [4-¹⁸F]fluoroglutamine via intravenous injection and imaged using a microPET scanner. Imaging was initiated 3 h post treatment with vehicle or V-9302 (75 mg/kg, i.p.). Dynamic data sets were acquired for 60 min, reconstructed using OSEM3D/MAP algorithm. Tracer uptake was quantified as % injected dose per gram of tissue (%ID/g). [1]
- Chronic combination study in liver cancer xenografts: BALB/c nude mice bearing SNU398 or MHCC97H xenografts (tumor volume ~50-100 mm³) were randomly assigned to 5 days/week treatment with vehicle, CB-839 (150 mg/kg, oral gavage, twice per day), V-9302 (30 mg/kg, i.p.), or combination for 20 or 15 days. Tumor volume calculated by modified ellipsoidal formula: volume = ½ (length × width²). [2]
- V-9302 treatment in orthotopic TNBC model: C57BL/6 mice bearing E0771 tumors (100 mm³, ~11 days after inoculation) were treated with V-9302 (50 mg/kg, i.p., daily for 5 days). Tumors were harvested for flow cytometry and immunohistochemistry. [3]

- In healthy mice, steady-state plasma concentrations of V-9302 were achieved at 4 h post-administration with a half-life of approximately 6 h. [1]
- Following a single acute V-9302 exposure (4 h), plasma glutamine levels were elevated by approximately 50%, while plasma glucose levels were unchanged. In mice chronically exposed to V-9302 over a 21-day regimen, plasma glutamine levels were slightly decreased. [1]

- In chronic exposure studies (21 days) in athymic nude mice bearing xenografts, no substantial weight loss was observed in V-9302-treated cohorts (75 mg/kg/day) relative to vehicle-treated controls. [1]
- Liver pathology was similar among mice chronically treated with either V-9302 or vehicle. [1]
- In combination studies with CB-839 in liver cancer xenografts (V-9302 30 mg/kg/day, CB-839 150 mg/kg/day), no body weight reduction was observed in any treatment group. [2]
- V-9302 did not affect the viability of activated CD8+ T cells or CD4+ T cells in vitro at 10 μM. [3]

[1]. Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacyin preclinical models. Nat Med. 2018 Feb;24(2):194-202.

[2]. A powerful drug combination strategy targeting glutamine addiction for the treatment of human liver cancer. Elife. 2020;9:e56749.

[3]. Selective glutamine metabolism inhibition in tumor cells improves antitumor T lymphocyte activity in triple-negative breast cancer. J Clin Invest. 2021;131(4):e140100.

- V-9302 is a competitive small molecule antagonist of ASCT2-mediated glutamine transport, with a chemical structure of 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid. [1]
- DARTS assay confirmed a stable V-9302-ASCT2 interaction, and ASCT1 was not stabilized, suggesting ASCT2 selectivity. [1]
- The combination of V-9302 with the GLS1 inhibitor CB-839 showed synergistic antitumor effects in glutamine-dependent liver cancer cells by depleting glutathione and inducing lethal ROS levels, which was rescued by NAC. [2]
- In TNBC models, V-9302 selectively blocked glutamine uptake by tumor cells but not CD8+ T cells, which adapted by upregulating the alternative glutamine transporter ATB⁰⁺/Slc6a14, leading to improved T cell effector function (increased GZMB, CD107a, IFN-γ) and decreased tumor growth. [3]
- The inverse correlation between glutamine metabolism gene signature and T cell cytotoxicity signature in human basal-like breast cancer was associated with poor patient survival. [3]

C34H39CLN2O4

575.137468576431

574.259

C, 71.00; H, 6.84; Cl, 6.16; N, 4.87; O, 11.13

2416138-42-4

V-9302;1855871-76-9

145710038

White to off-white solid powder

3

6

14

41

687

1

CC1=CC(=CC=C1)COC2=CC=CC=C2CN(CC[C@@H](C(=O)O)N)CC3=CC=CC=C3OCC4=CC=CC(=C4)C.Cl

LUQMUDMPRZSZTJ-YNMZEGNTSA-N

InChI=1S/C34H38N2O4.ClH/c1-25-9-7-11-27(19-25)23-39-32-15-5-3-13-29(32)21-36(18-17-31(35)34(37)38)22-30-14-4-6-16-33(30)40-24-28-12-8-10-26(2)20-28;/h3-16,19-20,31H,17-18,21-24,35H2,1-2H3,(H,37,38);1H/t31-;/m0./s1

(2S)-2-amino-4-[bis[[2-[(3-methylphenyl)methoxy]phenyl]methyl]amino]butanoic acid;hydrochloride

2416138-42-4; V-9302 hydrochloride; V9302 hydrochloride; V-9302 HCl; V-9302 (hydrochloride); (S)-2-Amino-4-(bis(2-((3-methylbenzyl)oxy)benzyl)amino)butanoic acid hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~100 mg/mL (~173.87 mM )
H2O :~50 mg/mL (~86.94 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)