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| Targets |
Mutant isocitrate dehydrogenase 1 (IDH1). GSK321 is a selective and potent mutant IDH1 inhibitor.
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| ln Vitro |
GSK321 has IC50 values of 4.6 nM, 3.8 nM, and 2.9 nM for R132H, R132C, and R132G, respectively, indicating that it is strongly suppressive to mutant IDH1 enzyme [1]. H3K9me2 levels are dramatically decreased by GSK321 (0, 0.5, 5 μM; 48 hours) [1]. GSK321 inhibits the growth of primary IDH1 mutant AML cells and decreases intracellular 2-HG [1]. GSK321 has inhibitory effect towards mutant IDH1, surmounting the typical differentiation barriers of AML cells and promoting myeloid differentiation of IDH1 mutant cells to the extent of leukemic blasts and stem cell-like cells [1]. In IDH1 mutant AML cells, GSK321 results in genome-wide DNA cytosine hypomethylation [1].
GSK321 inhibits IDH1 R132G, R132C, R132H, and wild-type IDH1 with IC50 values of 2.9, 3.8, 4.6, and 46 nM, respectively. It has >100-fold selectivity over IDH2. GSK321 induces intracellular 2-HG reduction and relieves myeloid differentiation blockade, inducing granulocytic differentiation at the level of leukemic blasts and more immature stem cells. GSK321 inhibits lipid synthesis and blocks cell proliferation in OPA1-deficient MEFs and prevents cytoplasmic glutamine reductive carboxylation. Treatment of primary R132G IDH1 mutant AML cells with GSK321 leads to a stable decrease of intracellular 2-HG levels for up to 22 days in suspension culture. GSK321 (0.1-10000 nM; 24 h) inhibits intracellular 2-HG production in HT1080 cells with an EC50 of 85 nM. |
| ln Vivo |
In vivo, GSK321 has been used in the study of leukemia. It induces myeloid differentiation and may have antitumor activity in acute myeloid leukemia (AML) models. The compound is orally bioavailable. Specific dosing regimens and detailed efficacy data are available in the primary literature but not extensively detailed in public search results.
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| Enzyme Assay |
IDH1 enzyme activity is assessed using in vitro biochemical assays measuring the conversion of isocitrate to α-ketoglutarate or the production of 2-hydroxyglutarate (2-HG). Recombinant wild-type or mutant IDH1 enzymes are incubated with isocitrate and NADP+, and product formation is measured by NADPH production (spectrophotometric) or by LC-MS for 2-HG quantification. IC50 values are calculated from dose-response curves using nonlinear regression analysis. Selectivity is evaluated by testing against wild-type IDH1 and IDH2.
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| Cell Assay |
Western Blot analysis [1]
Cell Types: HT-1080 Cell Tested Concentrations: 0, 0.5, 5 μM Incubation Duration: 48 h Experimental Results: diminished histone H3K9 dimethylation (H3K9me2). Cell proliferation assay[1] Cell Types: IDH1 mutant AML cells Tested Concentrations: 3 μM Incubation Duration: 15 days Experimental Results: There was an initial significant increase in cell number (2-fold to 15-fold) in IDH1 mutant AML cells. Cell cycle analysis[1] Cell Types: IDH1 Mutant AML Cell Tested Concentrations: Incubation Duration: 7 Days Experimental Results: A reproducible and significant reduction in quiescent (G0) phase cells was observed in R132G IDH1 and R132C IDH1 AML cells. Cellular activity is evaluated in IDH1 mutant cancer cell lines such as HT1080 (fibrosarcoma with IDH1 R132C mutation) and primary AML cells with IDH1 R132G mutations. Cells are treated with GSK321 at various concentrations (typically 0.1-10000 nM) for appropriate time periods (e.g., 24 hours for 2-HG measurement, 3-7 days for differentiation assays). Intracellular 2-HG levels are measured by LC-MS. Cell proliferation is assessed by MTT, CellTiter-Glo, or colony formation assays. Myeloid differentiation is assessed by flow cytometry for differentiation markers (CD11b, CD14, CD15) and by morphological examination of cytospin preparations. Lipid synthesis is measured by incorporation of radiolabeled acetate or by mass spectrometry-based lipidomics. Glutamine reductive carboxylation is assessed by measuring the incorporation of [U-13C]-glutamine into citrate and other metabolites by LC-MS. |
| Animal Protocol |
In vivo efficacy is studied in mouse xenograft models of IDH1 mutant leukemia or other cancers. GSK321 is administered orally at doses determined from pharmacokinetic and tolerability studies. Tumor growth is measured by caliper twice weekly (for solid tumors) or by measuring leukemia burden in blood, bone marrow, and spleen (for leukemia models). Intracellular 2-HG levels are measured in tumor tissue, blood, and bone marrow by LC-MS. Myeloid differentiation is assessed by flow cytometry for differentiation markers on leukemic blasts in blood and bone marrow. Pharmacodynamic markers include 2-HG levels, IDH1 target engagement, and differentiation status. The compound is orally bioavailable.
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| ADME/Pharmacokinetics |
GSK321 has a molecular formula of C22H23N5O3 and a molecular weight of 405.45 g/mol. CAS Number: 1816331-63-1. It is a selective and potent mutant IDH1 inhibitor. Purity: typically ≥98% by HPLC. The compound is orally bioavailable. Solubility: soluble in DMSO. Storage: store at -20°C for long-term stability, protected from light and moisture. Detailed pharmacokinetic parameters including half-life, oral bioavailability, volume of distribution, and plasma protein binding are available from the primary literature.
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| Toxicity/Toxicokinetics |
Safety data for GSK321 indicate it is a research compound for laboratory use only. As an IDH1 inhibitor, it may have effects on cellular metabolism, hematopoiesis, and differentiation. Specific toxicological profiles including acute toxicity, organ toxicity, genotoxicity, and reproductive toxicity are not extensively published. Standard laboratory safety practices should be followed during handling, including the use of personal protective equipment and adequate ventilation. The compound is for research use only and not for human therapeutic applications without appropriate regulatory approval. Consult the material safety data sheet (MSDS) for detailed safety information.
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| References |
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| Additional Infomation |
GSK321 is a research tool compound for studying IDH1 mutant biology and acute myeloid leukemia. It is not approved for clinical use. The compound has been investigated for AML and other cancers due to its ability to inhibit mutant IDH1, reduce 2-HG, and induce myeloid differentiation. GSK321 is a chiral, allosteric, and orally bioavailable small molecule. Reference: (S,R)-WT IDH1 Inhibitor 2 (GSK321) is a potent, selective mutant IDH1 inhibitor with IC50 values of 2.9, 3.8, 4.6, and 46 nM for R132G, R132C, R132H, and WT IDH1, respectively, and >100-fold selectivity over IDH2. The compound's ability to reduce 2-HG and induce differentiation makes it a valuable tool for validating IDH1 as a therapeutic target in AML and other IDH1-mutant cancers.
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| Molecular Formula |
C28H28FN5O3
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|---|---|
| Molecular Weight |
501.5520
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| Exact Mass |
501.217
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| CAS # |
1816331-63-1
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| Related CAS # |
(S,R)-GSK321;1816272-18-0
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| PubChem CID |
91864709
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| Appearance |
Off-white to light brown 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 |
FC1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])N1C2=C(C(C(N([H])C3=C([H])C([H])=C([H])C([C@]([H])(C([H])([H])[H])O[H])=C3[H])=O)=N1)C([H])([H])N(C(C1=C([H])C([H])=C([H])N1[H])=O)C([H])([H])[C@@]2([H])C([H])([H])[H]
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| InChi Key |
IVFDDVKCCBDPQZ-MSOLQXFVSA-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+/m1/s1
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| Chemical Name |
(7R)-1-[(4-fluorophenyl)methyl]-N-[3-[(1S)-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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| Synonyms |
GSK-321.; GSK 321; GSK321
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~250 mg/mL (~498.45 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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