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Purity: ≥98%
TCS-PIM-1-4a ( (the mixture of E- and Z-enantiomers of SMI-4a) is a novel Pim inhibitor with IC50 of 13 nM for Pim-1 and 2.3 μM for Pim-2. The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. TCS-PIM-1-4a was identified by screening to be a Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although TCS-PIM-1-4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases demonstrated that it has high selectivity for Pim kinases. Overall, TCS-PIM-1-4a represents a new Pim kinase inhibitor that may provide leads to novel anticancer agents.
| Targets |
PIM1/2
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| ln Vivo |
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| Enzyme Assay |
Pim protein kinase assays were conducted using multiple methods to ensure that the effects of the compounds were not due to any experimental artifacts. The primary screen and evaluation of the compounds shown in Table 3 was conducted using an ATP-depletion assay. Briefly, recombinant human Pim-1 was incubated with S6 kinase/Rsk-2 peptide 2 (KKRNRTLTK) as the substrate in the presence 100 µM of compounds from the screening library, 1 µM ATP and 10 mM MgCl2 for 1 h. The Kinase-Glo luciferase kit was used to measure residual ATP levels after the kinase reaction. For experiments that required higher ATP concentrations, Pim-1 kinase activity was monitored spectrophotometrically using a coupled assay in which ADP production is coupled to NADH oxidation catalyzed by pyruvate kinase and lactate dehydrogenase. Assays were carried out in 20 mM MOPS pH 7 containing 100 mM NaCl, 10 mM MgCl2, 2.5 mM phosphoenolpyruvate, 0.2 mM NADH, 30 µg/mL pyruvate kinase, 10 µg/mL lactate dehydrogenase, 2 mM dithiothreitol, 25 nM Pim-1, 100 µM S61 peptide, and varying concentrations of ATP. Activity was measured by monitoring NADH oxidation as the decrease at 340 nm in a VersaMax microplate reader (Molecular Devices) at 25 °C. Reactions were initiated by the addition of ATP (typically 100 µM). Inhibitors (final 1% DMSO) were added just prior to the addition of ATP. In either case, IC50 values were determined using nonlinear regression with the program GraphPad Prism. In some experiments, Pim-1 kinase activity was determined using His-tagged 4E-BP1 as the substrate. The active Pim-1 protein was resuspended in kinase reaction buffer (10 mM MOPS, pH 7.4, 100 µM ATP, 15 mM MgCl2, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT, and protease inhibitor cocktail). In each reaction (30 µL), 3 µg of His-4E-BP1 protein was used as substrate, and 10 μCi of [γ-32P] ATP were then added. Incubation was carried out at 30 °C for 30 min with agitation. The samples were then subjected to SDS-PAGE and 32P labeled 4E-BP1 was visualized by autoradiography. Finally, Pim-1 activity in intact cells was measured in some experiments. HEK-293T cells were transfected with Flag-Pim-1 for 24 h, and then were trypsined and divided into smaller dishes for overnight. Cells were washed once and incubated with phosphate-free media containing 10% phosphate-free FBS for 1 h. Cells were then incubated in medium containing 50 μCi/ml [32P]orthophosphate for 4 h, in which the test compounds were added for the final 1 h. To immunoprecipitate Pim-1, anti-Flag M2 agarose was added to the cell lysate and incubated for 3 h. A portion (10%) of the immunoprecipitates was used for Western blotting with anti-Flag antibodies (input). The other 90% of each sample was subjected to SDS-PAGE, and 32P-labeled Pim-1 was visualized by autoradiography.
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| Cell Assay |
Biochemical Analysis. K562 cells were transfected with scrambled siRNA or siPim-1 (ON-TARGETplus SMARTpool) using Lipofectamine™2000 according to the manufacturer’s protocol, and 48 h posttransfection lysates were prepared. Cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ATP, ADP, and AMP were measured by HPLC as described previously, and ATP was also measured using the ATP Bioluminescence Assay Kit HS II with 105 cells. eIF4E was captured on m7-GTP sepharose from WT and TKO MEFs lysate and bound 4EBP1 and eIF4G determined by Western blotting.[1]
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| References |
Proc Natl Acad Sci U S A.2011 Jan 11;108(2):528-33.;Mol Cancer Ther.2009 Jun;8(6):1473-83;Blood.2010 Jan 28;115(4):824-33.
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| Additional Infomation |
The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMPATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5'-cap-dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.[1]
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| Molecular Formula |
C11H6F3NO2S
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| Molecular Weight |
273.23
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| Exact Mass |
273.007
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| Elemental Analysis |
C, 48.36; H, 2.21; F, 20.86; N, 5.13; O, 11.71; S, 11.73
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| CAS # |
327033-36-3
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| Related CAS # |
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| PubChem CID |
1361334
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.602
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| LogP |
2.3
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
18
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| Complexity |
406
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(NC/1=O)SC1=C\C2=CC=CC(C(F)(F)F)=C2
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| InChi Key |
NGJLOFCOEOHFKQ-VMPITWQZSA-N
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| InChi Code |
InChI=1S/C11H6F3NO2S/c12-11(13,14)7-3-1-2-6(4-7)5-8-9(16)15-10(17)18-8/h1-5H,(H,15,16,17)/b8-5+
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| Chemical Name |
(5E)-5-[[3-(trifluoromethyl)phenyl]methylidene]-1,3-thiazolidine-2,4-dione
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| Synonyms |
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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 |
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| 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) |
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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 | 3.6599 mL | 18.2996 mL | 36.5992 mL | |
| 5 mM | 0.7320 mL | 3.6599 mL | 7.3198 mL | |
| 10 mM | 0.3660 mL | 1.8300 mL | 3.6599 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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