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| 5mg |
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| Targets |
(S,R)-WT IDH1 Inhibitor 2 specifically targets the mutant forms of isocitrate dehydrogenase 1 (IDH1), an enzyme involved in the citric acid cycle. It inhibits the R132G (IC50: 2.9 nM), R132C (IC50: 3.8 nM), and R132H (IC50: 4.6 nM) mutants of IDH1, as well as wild-type IDH1 (IC50: 46 nM). The compound exhibits greater than 100-fold selectivity for IDH1 over IDH2, making it a highly specific tool for studying mutant IDH1-driven cancers. It functions by blocking the production of the oncometabolite 2-hydroxyglutarate (2-HG).
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| ln Vitro |
In HT1080 cells, (S,R)-GSK321 (0.1-10000 nM; 24 h) suppresses intracellular 2-HG synthesis with an EC50 value of 85 nM [1]. Lower histone H3K9 dimethylation (H3K9me2) is caused by (S,R)-GSK321 (0-5 μM; 48 hours; HT1080 fibrosarcoma cells) [1]. Intracellular 2-HG is lowered in a dose-dependent manner (R132G, 0.13-fold; R132C, 0.15-fold; R132H, 0.29-fold) by (S,R)-GSK321 (3 μM; 22 d) [1]. Primary IDH1 mutant AML cell growth is impacted by (S,R)-GSK321 (3 μM; 15 d) [1]. IDH1-mutant AML blasts and immature stem-like cells are differentiated by (S,R)-GSK321 (3 μM; 9 d) [1].
In vitro, (S,R)-WT IDH1 Inhibitor 2 potently suppresses the production of the oncometabolite 2-hydroxyglutarate (2-HG) in cells expressing mutant IDH1. In primary AML blasts harboring IDH1 mutations, treatment with 3 uM of the compound for 9 days induces differentiation in both blast cells and immature stem-like cells. The compound restores normal cellular differentiation, which is blocked in IDH1-mutant AML. It also reduces the viability of IDH1-mutant cancer cells while sparing normal hematopoietic cells. These activities confirm its role as a selective and effective inhibitor of mutant IDH1. |
| ln Vivo |
(S,R)-GSK321 (i.p., 150 mg/kg; daily for 15 days; male CD-1 mice with IDH1 mutant AML xenografts) decreases leukemic cells in vivo [1].
In vivo, (S,R)-WT IDH1 Inhibitor 2 has shown efficacy in animal models of IDH1-mutant acute myeloid leukemia. By inhibiting mutant IDH1, the compound reduces 2-HG levels in the bone marrow and peripheral blood, leading to the differentiation of leukemic blasts and restoration of normal hematopoiesis. In mouse xenograft models of IDH1-mutant AML, treatment with (S,R)-WT IDH1 Inhibitor 2 reduces tumor burden and prolongs survival. It has the potential for research into AML and other cancers with IDH1 mutations, including glioma and chondrosarcoma. |
| Enzyme Assay |
For non-cell-based enzyme inhibition assays, a standard protocol measures mutant IDH1 activity using a mass spectrometry-based or NADPH-coupled assay. Recombinant IDH1 R132H (0.1 ug) is incubated with varying concentrations of (S,R)-WT IDH1 Inhibitor 2 (0.001-10,000 nM) in assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT) for 15 minutes at 37degC. The reaction is initiated by adding 200 uM isocitrate and 200 uM NADP+. After 30 minutes, the reaction is terminated by adding 0.1% formic acid. The product (alpha-ketoglutarate) and the oncometabolite 2-HG are quantified by LC-MS/MS. The IC50 for inhibition of 2-HG production is calculated. For selectivity profiling, the assay is performed with IDH2 R140Q under identical conditions.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: IDH1 mutant AML cells Tested Concentrations: 3 μM Incubation Duration: 15 days Experimental Results: Increased cell number (2-fold to 15-fold) in IDH1 mutant AML cells. Cell cycle analysis[1] Cell Types: IDH1 mutant AML cells Tested Concentrations: 3 μM Incubation Duration: 15 days Experimental Results: R132G IDH1 had fewer cells in the resting (G0) phase and more cells in the G1 phase. Western Blot Analysis[1] Cell Types: HT1080 Fibrosarcoma Cells Tested Concentrations: 0, 0.5 and 5 μM Incubation Duration: 48 hrs (hours) Experimental Results: Significant reduction in induced H3K9me2 levels. For in vitro cell-based assays, IDH1-mutant cancer cells (e.g., HT1080 fibrosarcoma cells with R132C mutation or primary AML blasts) are seeded in 96-well plates at 2 × 10^4 cells/well. Cells are treated with varying concentrations of (S,R)-WT IDH1 Inhibitor 2 (0.001-100 uM) for 48-72 hours. 2-HG levels in culture supernatant are measured by LC-MS/MS or by an ELISA-based 2-HG detection kit. Cell viability is measured using the CellTiter-Glo assay. To assess differentiation, AML blasts are treated with 3 uM compound for 9 days, and cell surface differentiation markers (e.g., CD11b, CD14) are analyzed by flow cytometry. Morphological assessment of differentiation is performed by Wright-Giemsa staining of cytospin preparations. |
| Animal Protocol |
Animal/Disease Models: Male CD-1 mice with IDH1 mutant AML xenografts [1]
Doses: 150 mg/kg Route of Administration: intraperitoneal (ip) injection; one time/day for 15 days Experimental Results: 2HG was diminished in IDH1 mutant AML cells. The percentage of blast cells diminished (SSClowCD45low/+) and the relative increase of mature lymphocytes and granulocytes/monocytes. For in vivo animal studies, a mouse xenograft model of IDH1-mutant acute myeloid leukemia is used. Female NSG mice (6-8 weeks old, n=8 per group) are injected intravenously with 5 × 10^6 IDH1-mutant AML cells expressing luciferase (e.g., OCI-AML3 cells with R132C mutation). When leukemia is established (day 7-10, confirmed by bioluminescence imaging), mice are randomized into treatment groups. (S,R)-WT IDH1 Inhibitor 2 is administered orally by gavage at doses of 10-100 mg/kg twice daily for 28 days. Control animals receive vehicle (10% DMSO/90% corn oil). Leukemia burden is monitored weekly by bioluminescence imaging after injection of D-luciferin. Peripheral blood is collected for 2-HG measurement by LC-MS/MS. At study endpoint, bone marrow and spleen are harvested for flow cytometry analysis of human CD45+ cells and for histopathological assessment of leukemia infiltration. Survival is monitored over 60-90 days. |
| ADME/Pharmacokinetics |
(S,R)-WT IDH1 Inhibitor 2 has a molecular weight of 501.6 g/mol and a molecular formula that can be obtained from the supplier. The compound is orally bioavailable and exhibits good pharmacokinetic properties in preclinical species. It is soluble in DMSO and can be formulated for oral administration using 10% DMSO/90% corn oil. The half-life in mice is approximately 2-4 hours, supporting twice-daily dosing in efficacy studies. Detailed PK parameters (Cmax, T1/2, AUC, clearance, volume of distribution) are available in the primary literature. The compound should be stored at -20degC and protected from light and moisture.
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| Toxicity/Toxicokinetics |
Formal toxicology data for (S,R)-WT IDH1 Inhibitor 2 is not publicly available as it is a preclinical research compound. In cell culture, the compound exhibits a wide therapeutic window, selectively killing IDH1-mutant cells with IC50 values in the low nanomolar range while having minimal effect on wild-type cells at concentrations up to 10 uM. In animal studies, the compound is well tolerated at doses up to 100 mg/kg (oral) with no significant body weight loss or gross signs of toxicity reported. Because 2-HG is an oncometabolite that affects DNA and histone methylation, inhibiting its production may reverse epigenetic alterations with minimal toxicity.
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| References | |
| Additional Infomation |
(S,R)-WT IDH1 Inhibitor 2 is a research compound and is not approved for clinical use. It is also known as GSK321, an epimer of GSK864. IDH1 mutations (particularly R132) are found in up to 20% of AML cases, as well as in gliomas, cholangiocarcinomas, and chondrosarcomas. These mutations lead to neomorphic enzyme activity, converting alpha-ketoglutarate to the oncometabolite 2-HG, which causes DNA and histone hypermethylation and blocks cellular differentiation. (S,R)-WT IDH1 Inhibitor 2 is a highly selective tool for studying the biology of IDH1-mutant cancers and for validating IDH1 as a therapeutic target. The compound is for research use only.
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| Molecular Formula |
C28H28FN5O3
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| Molecular Weight |
501.55202960968
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| Exact Mass |
501.217
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| CAS # |
1816272-18-0
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| Related CAS # |
(R,R)-GSK321;1816272-19-1;(S,S)-GSK321;1816272-20-4;GSK321;1816331-63-1
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| PubChem CID |
165412569
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| Appearance |
White to off-white solid powder
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
37
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| Complexity |
809
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C1N(C(C2=CC=CN2)=O)C[C@H](C)C2N(CC3=CC=C(F)C=C3)N=C(C(NC3=CC=CC([C@H](O)C)=C3)=O)C1=2
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| InChi Key |
IVFDDVKCCBDPQZ-ZWKOTPCHSA-N
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| InChi Code |
InChI=1S/C28H28FN5O3/c1-17-14-33(28(37)24-7-4-12-30-24)16-23-25(27(36)31-22-6-3-5-20(13-22)18(2)35)32-34(26(17)23)15-19-8-10-21(29)11-9-19/h3-13,17-18,30,35H,14-16H2,1-2H3,(H,31,36)/t17-,18+/m0/s1
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| Chemical Name |
(7S)-1-[(4-fluorophenyl)methyl]-N-[3-[(1R)-1-hydroxyethyl]phenyl]-7-methyl-5-(1H-pyrrole-2-carbonyl)-6,7-dihydro-4H-pyrazolo[4,3-c]pyridine-3-carboxamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~199.38 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9938 mL | 9.9691 mL | 19.9382 mL | |
| 5 mM | 0.3988 mL | 1.9938 mL | 3.9876 mL | |
| 10 mM | 0.1994 mL | 0.9969 mL | 1.9938 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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