γ-Glutamylcysteine synthetase (γ-GCS) / Glutamate-cysteine ligase (GCL) - catalytic subunit (GCLC). IC50 (cell-free enzymatic assay) = 570 nM (95% CI: 429-757 nM). [1][3]

- BSO inhibited GCL activity in a cell-free enzymatic assay with an IC50 of 570 nM (95% CI: 429-757 nM). [3]
- In SH-SY5Y cells, BSO (0-500 μM) showed 80-85% cell viability at 250 μM by MTT assay, with no significant increase in apoptosis by Annexin-V/PI staining at 100 and 250 μM for 48 h. [1]
- In ZAZ and M14 melanoma cell lines, BSO (50 μM, 96 h) depleted GSH levels by approximately 95%. At lower concentrations (1-3 μM), GSH depletion was incomplete with partial recovery between 24-72 h. [1]
- In ZAZ and M14 cells, BSO (50 μM, 48-96 h) decreased GST enzyme activity by approximately 2-3 fold. Western blot analysis showed decreased GST-μ protein levels, while GST-π expression was unaffected. Northern blot analysis confirmed reduced GST-μ mRNA levels. [1]
- BSO (50 μM, 48-96 h) decreased DNA synthesis (³H-TdR incorporation) in ZAZ and M14 cells. Cytotoxic effects (60% decrease in cell counts at 96 h) were observed in ZAZ cells, while M14 cells showed cytostatic effects (stable cell numbers at 48-72 h with 2-fold increase at 96 h). [1]
- In PANC-1 pancreatic cancer cells, BSO (100 μM, 24 h) reduced total glutathione (GSH+GSSG) levels and induced lipid peroxidation (measured by BODIPY 581/591 C11 staining). The viability-reducing effect was attenuated by ferrostatin-1 (ferroptosis inhibitor), GSH monoethyl ester (GSHee), and N-acetylcysteine (NAC), and enhanced by ferric ammonium citrate (FAC). [3]
- In SW48 colon cancer cells, BSO-induced viability reduction was attenuated by NAC but not by ferrostatin-1, suggesting a ferroptosis-independent mechanism. [3]
- In a panel of cancer cell lines (colorectal, kidney, pancreatic, ovarian, prostate), BSO showed variable sensitivity. Sensitive cell lines (logIC50 < -4.0 M) included G402 (kidney, -5.73), PANC-1 (pancreas, -5.33), RCC4 VHL-/- (kidney, -4.777), 786-O (kidney, -4.10), A-498 (kidney, -4.09), A2780 CDDP (ovary, -4.05), and SW48 (colon, -4.04). Resistant cell lines (logIC50 > -3.5 M) included HCT-15, SW620, COLO 205, LS 174T, HCT-116, RKO, HT-29, SW480, ACHN, and DU 145. [3]
- Basal total glutathione levels were lower in BSO-sensitive cells (G402, RCC4 VHL-/-, A-498) compared to insensitive cells (RCC4 VHL+/+, Caki-2, HCT-116; P=0.08). GCLC protein levels positively correlated with glutathione levels (r²=0.814, P=0.04), while GSS protein levels showed no correlation (r²=0.021, P=0.82). [3]

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In ZAZ and M14 melanoma cell lines, 48 hours of treatment with L-buthionine-(S,R)-suLfoximine (BSO: 50 μM) led to a 95% reduction in GSH levels and a 60% reduction in GST enzyme activity. The cellular oxidative intermediate g-glutamine is induced in different cell lines by GST-π L-butyrine-(S,R)-suLfoximine (BSO) through irreversibly blocking g-glutamine cysteine synthetase Amide protein and mRNA levels were drastically reduced [1]. An enzyme called semiphosphatidylamine synthetase is necessary for the production of glutathione (GSH) [2]. Ferroptosis is induced by L-butanthionine-(S,R)-suLfoximine (BSO) [3].

- In C57BL/6J pᵘⁿ/pᵘⁿ mice, administration of BSO via drinking water (2 mM or 20 mM) from 0.5 to 18.5 days post-coitum resulted in a dose-dependent increase in the frequency of 70 kb DNA deletions (eye-spots in retinal pigment epithelium). 2 mM BSO caused ~30% more eye-spots (7.79 ± 0.45 vs. 5.36 ± 0.29 in controls, P<0.001) and 20 mM BSO caused ~40% more eye-spots (8.78 ± 0.61 vs. 5.36 ± 0.29, P<0.001). Co-treatment with 20 mM NAC normalized the deletion frequency (6.18 ± 0.47 vs. 7.79 ± 0.45, P=0.016). [2]
- In the same mouse model, BSO treatment (2 mM and 20 mM) reduced GSH concentrations in fetuses by 55% (P<0.01) and 70% (P<0.001), respectively, and reduced cysteine concentrations by 27% (P<0.05) and 55% (P<0.01), respectively. NAC co-treatment restored cysteine levels but not GSH levels. [2]
- In xenograft studies, BSO treatment prolonged survival of melanoma-bearing mice (reference to Prezioso et al., 1990b, 1992). [1]
- In a phase I clinical trial, continuous infusion of BSO achieved steady-state serum concentrations exceeding 500 μM and depleted tumor glutathione to <10% of baseline levels. One patient with metastatic malignant melanoma sustained a complete response after treatment with continuous infusion BSO plus bolus melphalan. [1]

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In developing mice, BSO raises the frequency of DNA. In comparison to animals that were not treated, BSO therapy decreased GSH levels in mice that were given 2 mM and 20 mM BSO, respectively, by 55% and 70%. GSH was outpaced by co-treatment with 2 mM BSO and 20 mM NAC. The degree is comparable to that of 2 mM BSO, which is in line with BSO's ability to inhibit the g-GCS enzyme, which is responsible for the synthesis of dyes from GSH. C57BL/6J pun/pun mice are an animal model for cysteine modules, similar to GSH, following BSO therapy [2].

- Cell-free GCL enzymatic assay: N-terminal His-tagged human GCLM and C-terminal His-tagged human GCLC were expressed in E. coli and purified by Ni-NTA affinity chromatography followed by Superdex 200 gel filtration. BSO (0.1, 1, 10, and 100 μM) was premixed with enzymes (10 nM each) for 30 min prior to addition of 200 μM ATP, 1.2 mM glutamic acid, and 200 μM cysteine. After 60 min incubation, the reaction was terminated with 1% formic acid, and ATP and γ-glutamylcysteine levels were measured using RapidFire300 mass spectrometry coupled with an API4000 triple quadrupole mass spectrometer. IC50 was calculated using XLfit or GraphPad Prism. [3]
- GST enzyme activity assay: Cell supernatants were assayed for glutathione S-transferase activity using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates according to the method of Habig et al. (1975). Activity was measured spectrophotometrically. [1]
- Glutathione reductase (GSSG-R) assay: Activity was assayed by following the rate of NADPH oxidation spectrophotometrically. [1]
- Glutathione peroxidase (GSH-Px) assay: Activity was measured by the method of Paglia and Valentine using H₂O₂ as substrate. [1]

- Agar-based thymidine incorporation assay: Tumor cells were suspended in soft agar and plated in 24-well plates at 20,000 cells per well for 5 days with or without BSO. For combination experiments, cells were preincubated in BSO for 24 h. Wells were pulsed with ³H-thymidine for the last 48 h. Plates were heated to 90°C to liquefy agar, cells harvested onto glass fiber filters, and radioactivity counted. Fraction of control (FC) proliferation = treated CPM/control CPM. [1]
- Cell viability assay (CellTiter-Glo): Cells were seeded at 1,000-3,000 cells/well in 96-well plates. After 24 h, BSO, GSHee, ferrostatin-1, NAC, cisplatin, or FAC were added. After 3 days, cell viability was assessed using CellTiter-Glo Luminescent Cell Viability Assay. [3]
- Total glutathione measurement (GSH+GSSG): Cellular total glutathione levels were determined using GSH/GSSG-Glo Assay (Promega) after 24 h incubation with BSO. [3]
- Lipid peroxidation measurement: PANC-1 cells (1 × 10⁶) were seeded in 10-cm dishes, treated with BSO for 24 h, then incubated with 5 μM BODIPY 581/591 C11 Lipid Peroxidation Sensor for 30 min. After washing, lipid peroxidation was assessed by flow cytometry (FACS Verse). [3]
- GSH assay (modified Tietze method): Cells were freeze-thawed three times in 0.01 M NaPO₄ + 0.005 M EDTA buffer (pH 7.5), centrifuged at 30,000g for 30 min at 4°C. GSH levels were determined from the rate of change in optical density at 412 nm at 25°C. [1]
- Western blotting: Cells were lysed in SDS sample buffer, heated at 95°C for 5 min. Proteins (3 μg) were separated by SDS-PAGE (7.5-15% gradient gel), transferred to PVDF membranes, blocked, and probed with primary antibodies (anti-GCLC, anti-GSS, anti-Hsp90) overnight at 4°C, followed by HRP-labeled secondary antibodies. Chemiluminescence was detected using LAS-3000. [3]
- Northern blotting: Total RNA (50 μg) was electrophoresed on agarose-formaldehyde gel, transferred to nitrocellulose paper, and hybridized at 65°C with ³²P-random primer labeled cDNA probes for GST-μ and GST-π. [1]
- Immunohistochemistry (GST-π): Paraffin-embedded tissue sections were deparaffinized, incubated with 3% H₂O₂, blocked, incubated with anti-GST-π monoclonal antibody (1:10) for 60 min, then with LINK and LABEL reagents, exposed to DAB, counterstained with hematoxylin. Percentage of stained cells and intensity were determined by light microscopy. [1]

Oral administration in drinking water (mouse pregnancy study): Pregnant C57BL/6J pᵘⁿ/pᵘⁿ mice were given free access to drinking water supplemented with BSO (2 mM or 20 mM), alone or in combination with 20 mM NAC, from 0.5 to 18.5 days post-coitum. Daily BSO intake was approximately 0.1 g/kg (0.45 mM/kg) at 2 mM or 1 g/kg (4.5 mM/kg) at 20 mM. Offspring were sacrificed at 20 days of age for eye-spot analysis, or fetuses were isolated at 17.5 d.p.c. for thiol determination. [2]
- Intraperitoneal administration in mice: C57BL/6J mice were injected i.p. with BSO (300 mg/kg) once daily for 7 days, alone or preceded by D-mannitol (2.0 M) for BBB disruption. [1]
- Intravenous administration in mice: C57BL/6J or BALB/cByJ mice were injected i.v. via tail vein with BSO at various doses (equivalent to 75, 115, 150, 300 mg/kg of compound 1) once daily for 3 or 7 days. [1]
- Xenograft studies (referenced): Melanoma-bearing mice were treated with BSO alone or in combination with other agents. [1]

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Animal/Disease Models: C57BL/6J pun/pun mice[2].
Doses: 2 mM L-Buthionine-(S,R)-sulfoximine (BSO), 20 mM BSO, 2 mM BSO and 20 mM NAC, 20 mM NAC or unsupplemented water for 18 days from 0.5 to 18.5 d.p.c. The pH of supplemented water is as follows: 6.88, 20 mM BSO; 3.37, 2 mM BSO; 2.65, 2 mM BSO plus 20 mM NAC; and 2.58, 20 mM NAC. The pH of regular water used in our facility is ~4.
Route of Administration: Drinking water
Experimental Results: The average number of eye-spots (mean±SEM) is 5.36±0.29 (n=46), 7.79±0.45 (n=34) and 8.78±0.61 (n=32) in untreated controls, 2 mM L-Buthionine-(S,R)-sulfoximine (BSO) and 20 mM BSO treated mice, respectively. The 2 mM BSO treatment results in ~30% more eye-spots, and the 20 mM treatment results in 40% more eye-spots compared with untreated mice.

- In phase I clinical trials, continuous infusion of BSO achieved steady-state serum concentrations exceeding 500 μM. [1]
- Continuous infusion BSO depleted tumor glutathione to less than 10% of baseline levels. [1]
- The area under the curve (AUC) for BSO in vitro at the IC90 was 3,060 μM × hr, compared to 42,192 μM × hr in vivo (13.8-fold higher in vivo). [1]
- BSO is stable during 5-day incubation in culture media. [1]

- In phase I clinical trials, BSO administered by intravenous bolus and by continuous infusion was well tolerated. [1]
- In pregnant rats, BSO doses of 2-6 mM/kg/day in drinking water throughout pregnancy lowered GSH levels but had no teratogenic effects in offspring. [2]
- In mouse fetuses, BSO treatment (2 mM and 20 mM in drinking water) caused significant GSH and cysteine depletion, but co-treatment with NAC prevented DNA deletions without restoring GSH levels. [2]
- In normal mice, histopathological analysis of liver sections after BSO treatment (0.26 mmol/kg, i.v. once daily for 3 days) showed no visible changes compared to controls. Serum creatinine and liver enzyme levels (ALT, AST) remained within standard ranges. [1]
- BSO administration to newborn rats causes multiorgan failure and death. [2]

[1]. Selective and synergistic activity of L-S,R-buthionine sulfoximine on malignant melanoma is accompanied by decreased expression of glutathione-S-transferase. Pigment Cell Res. 1997 Aug;10(4):236-49.

[2]. Glutathione depletion by buthionine sulfoximine induces DNA deletions in mice. Carcinogenesis. 2006 Feb;27(2):240-4.

[3]. Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors. Oncol Lett. 2018 Jun;15(6):8735-8743.

BSO selectively inhibits GSH synthesis by irreversibly inhibiting γ-glutamylcysteine synthetase (γ-GCS) via covalent linkage of the L-buthionine-S-sulfoximine isomer to the active site. [1][2]
- Melanoma cells are uniquely sensitive to BSO due to their dependence on GSH for detoxifying reactive orthoquinones and peroxides produced during melanin synthesis. BSO sensitivity of melanoma specimens correlated with melanin content (r=0.63). [1]
- BSO synergistically enhanced the cytotoxicity of BCNU (carmustine) against melanoma cell lines and fresh human melanoma specimens. The combination index (CI) was 0.7 at 1 μM BSO and 0.57 at 3 μM BSO. This synergy may be partly due to BSO-mediated downregulation of GST-μ expression. [1]
- BSO treatment led to decreased GST enzyme activity and selective downregulation of GST-μ (but not GST-π) at both protein and mRNA levels. [1]
- BSO induces ferroptosis in some cancer cells (e.g., PANC-1), characterized by iron-dependent lipid peroxidation and attenuation by ferrostatin-1. However, in other cells (e.g., SW48), BSO-induced cell death appears ferroptosis-independent. [3]
- GST-π positive melanoma specimens were approximately 2-fold more resistant to BSO (P<0.001) and 2.5-fold more resistant to the BSO-BCNU combination (P<0.02) than GST-π negative specimens. [1]
- In colorectal cancer patient samples, approximately 15% (44/284) of tumors exhibited lower glutathione levels compared to matched normal tissues, suggesting a potential patient population that may benefit from GCL inhibitor therapy. [3]

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L-Butylthionine-(S,R)-sulfonylimide is a 2-amino-4-(S-butylsulfonylimide acyl)butyric acid with an S-configuration. It is an inhibitor of γ-glutamylcysteine synthase and glutathione (GSH) biosynthesis, and can enhance the apoptotic effects of various chemotherapeutic agents. It functions as a ferroptosis inducer and an EC 6.3.2.2 (glutamate-cysteine ligase) inhibitor. Butylthionine-sulfonylimide is a synthetic amino acid. It irreversibly inhibits γ-glutamylcysteine synthase, thereby depleting intracellular glutathione. Glutathione is a metabolite that plays a crucial role in protecting cells from oxidative stress, and inhibition of butylthionine-sulfonylimide leads to free radical-induced apoptosis. Elevated glutathione levels are associated with tumor cell resistance to alkylating agents and platinum compounds. By depleting intracellular glutathione, this drug may enhance the in vitro and in vivo cytotoxicity of various chemotherapeutic agents against drug-resistant tumors. Butylthionine sulfoxide imine may also possess anti-angiogenic activity. (NCI04)

C8H18N2O3S

222.3051

222.103

C, 43.22; H, 8.16; N, 12.60; O, 21.59; S, 14.42

83730-53-4

DL-Buthionine-(S,R)-sulfoximine;5072-26-4;L-Buthionine-(S,R)-sulfoximine hydrochloride

119565

White to off-white solid powder

1.3±0.1 g/cm3

382.3±52.0 °C at 760 mmHg

224-228ºC (dec.)

185.0±30.7 °C

0.0±1.9 mmHg at 25°C

1.538

0.22

3

5

7

14

284

1

CCCCS(=N)(=O)CC[C@@H](C(=O)O)N

KJQFBVYMGADDTQ-CVSPRKDYSA-N

InChI=1S/C8H18N2O3S/c1-2-3-5-14(10,13)6-4-7(9)8(11)12/h7,10H,2-6,9H2,1H3,(H,11,12)/t7-,14?/m0/s1

(2S)-2-amino-4-(butylsulfonimidoyl)butanoic acid

L-Buthionine-(S,R)-sulfoximine; L-Buthionine sulfoximine, BSO; l-Buthionine-sulfoximine ; (2S)-2-Amino-4-(butylsulfonimidoyl)butanoic acid; (2S)-2-Amino-4-(S-butylsulfonimidoyl)butanoic Acid; L-BSO; 83730-53-4;

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)

H2O : ~18.33 mg/mL (~82.45 mM)
DMSO :< 1 mg/mL

Solubility in Formulation 1: 100 mg/mL (449.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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