Disodium (R)-2-Hydroxyglutarate

Catalog No.S7873 Batch:S787301

Technical Data

Formula	C₅H₆Na₂O₅
Molecular Weight	192.08	CAS No.	103404-90-6
Solubility (25°C)*	In vitro	Water	38 mg/mL (197.83 mM)
		DMSO	Insoluble
		Ethanol	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

Disodium (R)-2-Hydroxyglutarate (D-α-Hydroxyglutaric acid disodium) is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases with K_i of 10.87 ± 1.85 mM.

Targets

α-ketoglutarate-dependent dioxygenase ^[1]

In vitro

In U-87MG cells, (R)-2-Hydroxyglutarate acts as weak antagonists of α-KG to inhibit α-KG-dependent histone demethylases and increases dimethylation on both H3K9 and H3K79. ^[1] Besides, (R)-2-Hydroxyglutarate inhibits ATP synthase and mTOR signaling, and thus causes growth arrest and tumor cell killing. ^[2]

Protocol (from reference)

Kinase Assay:^{^[1]}	Enzymatic Assays To assay human JHDM1A/KDM2A demethylase activity toward H3K36me2, His tagged JHDM1A is first obtained by transforming pET28a-JHDM1A into Escherichia coli BL21 and protein expression is induced by addition of 1 mM IPTG at 30° C when cell density reaches 0.5 OD600 units. Cells are lysed by sonication and Ni-NTA agarose is used to purify His-JHDM1A fusion proteins. Histone demethylase assay is carried out by incubating 2 μg oligonucleosomes, 4 μg purified His-JHDM1A, and/or 10–50 mM L- or D-2-HG in histone demethylation buffer [50 mM HEPES (pH 8.0), 625 μM Fe(NH4)₂(SO₄)₂, 0.1–0.5 mM α-KG, 2 mM ascorbate] at 37° C for 2–3 hr and the reactions are stopped by the addition of SDS loading buffer and subsequently analyzed by western blotting using anti-H3K36me2 antibody. To measure CeKDM7A demethylase activity toward H3K9me2 and H3K27me2, two synthetic dimethylated peptides H3K9me2 [ARTKQTARK (me2)STGGKA] and H3K27me2 [QLATKAARK (me2)SAPAS] are used as substrates. Demethylase assays are carried out in the presence of 10 μg enzyme, 1 μg peptide in 20 μl buffer 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 μM Fe(NH4)₂(SO₄)₂2, 100 μM α-KG, 2 mM Vc, 10 mM PMSF for 3 hr. The demethylation reaction mixture is desalted by passing through a C18 ZipTip. To examine the inhibitory effect of 2-HG, various concentrations of 2-HG are incubated with KDM7A briefly before adding other reaction mixtures. The samples are analyzed by a MALDI-TOF/TOF mass spectrometer.
Cell Assay:^{^[2]}	Cell lines U87/IDH1(R132H), Octyl α-KG- or octyl 2-HG-treated U87, and HCT 116 IDH1(R132H/+) cells Concentrations ~800 μM Incubation Time 4 days Method Cells are seeded in 12-well plates and, after overnight incubation, treated with the indicated concentrations of each compound. After harvesting, cells are stained with acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI). Cell number and viability are measured based on AO and DAPI fluorescence as measured by NC3000 following the manufacturer’s instructions.

Kinase Assay:^{^[1]}

Enzymatic Assays
To assay human JHDM1A/KDM2A demethylase activity toward H3K36me2, His tagged JHDM1A is first obtained by transforming pET28a-JHDM1A into Escherichia coli BL21 and protein expression is induced by addition of 1 mM IPTG at 30° C when cell density reaches 0.5 OD600 units. Cells are lysed by sonication and Ni-NTA agarose is used to purify His-JHDM1A fusion proteins. Histone demethylase assay is carried out by incubating 2 μg oligonucleosomes, 4 μg purified His-JHDM1A, and/or 10–50 mM L- or D-2-HG in histone demethylation buffer [50 mM HEPES (pH 8.0), 625 μM Fe(NH4)₂(SO₄)₂, 0.1–0.5 mM α-KG, 2 mM ascorbate] at 37° C for 2–3 hr and the reactions are stopped by the addition of SDS loading buffer and subsequently analyzed by western blotting using anti-H3K36me2 antibody. To measure CeKDM7A demethylase activity toward H3K9me2 and H3K27me2, two synthetic dimethylated peptides H3K9me2 [ARTKQTARK (me2)STGGKA] and H3K27me2 [QLATKAARK (me2)SAPAS] are used as substrates. Demethylase assays are carried out in the presence of 10 μg enzyme, 1 μg peptide in 20 μl buffer 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 μM Fe(NH4)₂(SO₄)₂2, 100 μM α-KG, 2 mM Vc, 10 mM PMSF for 3 hr. The demethylation reaction mixture is desalted by passing through a C18 ZipTip. To examine the inhibitory effect of 2-HG, various concentrations of 2-HG are incubated with KDM7A briefly before adding other reaction mixtures. The samples are analyzed by a MALDI-TOF/TOF mass spectrometer.

Cell Assay:^{^[2]}

Cell lines
U87/IDH1(R132H), Octyl α-KG- or octyl 2-HG-treated U87, and HCT 116 IDH1(R132H/+) cells
Concentrations
~800 μM
Incubation Time
4 days
Method
Cells are seeded in 12-well plates and, after overnight incubation, treated with the indicated concentrations of each compound. After harvesting, cells are stained with acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI). Cell number and viability are measured based on AO and DAPI fluorescence as measured by NC3000 following the manufacturer’s instructions.

References

Selleck's Disodium (R)-2-Hydroxyglutarate has been cited by 7 publications

Itaconate ameliorates autoimmunity by modulating T cell imbalance via metabolic and epigenetic reprogramming [ Nat Commun, 2023, 14(1):984]	PubMed: 36849508
Induction of functional neutrophils from mouse fibroblasts by thymidine through enhancement of Tet3 activity [ Cell Mol Immunol, 2022, 10.1038/s41423-022-00842-9]	PubMed: 35301470
D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment [ Mol Ther, 2022, S1525-0016(22)00007-7]	PubMed: 35007759
NAD+ Metabolism Maintains Inducible PD-L1 Expression to Drive Tumor Immune Evasion [ Cell Metab, 2021, 33(1):110-127.e5]	PubMed: 33171124
Wild-Type IDH1 and Mutant IDH1 Opposingly Regulate Podoplanin Expression in Glioma [ Transl Oncol, 2020, 13(4):100758]	PubMed: 32208352
Isocitrate dehydrogenase1 mutation reduces the pericyte coverage of microvessels in astrocytic tumours [ J Neurooncol, 2019, 143(2):187-196]	PubMed: 31004262
Cytokine correlation analysis based on drug perturbation [Wallner FK, et al. Cytokine, 2016, 90:73-79]	PubMed: 27816795

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SHIPPING AND STORAGE
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