PIM1/2
Pim-1 protein kinase (competitive with ATP). IC50 = 17 ± 7 nM (using peptide substrate RSRHSSYPAGT corresponding to Bad amino acids 107-117). [1]
Pim-2 (inhibits). [1] (reference 20 indicates dual inhibition)
Also shows weak inhibition of DYRK1a (only 16a, not 4a). [1]

SMI-4a is a novel benzylidene-thiazolidine-2, 4-dione small molecule, potent and selective inhibitor of Pim1 with IC50 of 17 nM, it is modestly potent to Pim-2, and does not significantly inhibit any other serine/threonine- or tyrosine-kinases. SMI-4a blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2). The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. SMI-4a kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-LBL/T-ALL) being highly sensitive. Incubation of pre-T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27(Kip1), apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre-T-LBL cells. In immunodeficient mice carrying subcutaneous pre-T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. These data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL. SMI-4a is an ATP competitive inhibitor of Pim1 with IC50 of 17 nM. SMI-4a shows high selectivity for Pim1 against a panel of kinases. SMI-4a inhibits the in vitro phosphorylation by Pim-1 of the known substrate, the translational repressor 4E-BP1. SMI-4a (5μM) inhibits pancreatic and leukemic cells growth. SMI-4a reduces phosphorylation of the Pim target Bad in prostate and hematopoietic cells. SMI-4a causes cell cycle arrest and reverses the antiapoptotic activity of Pim-1. SMI-4a increases the amount of p27Kip1 in the nucleus. SMI-4a treatment of pre-T-LBL inhibits the mTOR pathway. SMI-4a reduces MYC protein expression in pre-T-LBL. SMI-4a treatment induces up-regulation of MAPK pathway

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Kinase Assay: SMI-4a is a selective ATP-competitive Pim-1 kinase inhibitor with an IC50 of 21 nM for Pim-1 compared to an IC50 of 100 nM for Pim-2 and with little or no activity against a panel of 50 other kinases tested.

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Cell Assay: 6812/2 cells were incubated for 24 hours and Jurkat cells, for 48 hours with SMI-4a (10μM) or dimethyl sulfoxide (DMSO) in serum-free medium. After incubation, cells were harvested, washed once in phosphate-buffered saline (PBS), fixed in cold 70% ethanol for 45 minutes, stained with propidium iodide solution that contains RNaseA for 30 minutes, and analyzed by flow cytometry.

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In K562 human erythroleukemia cells, treatment with SMI-4a (5 μM for 1 hour in serum-free medium) induced activation of AMPK as determined by increased phosphorylation of AMPKα at Thr172, as well as increased phosphorylation of the AMPK targets acetyl-CoA carboxylase (ACC) at Ser79 and raptor at Ser792. Concomitantly, SMI-4a inhibited mTORC1 activity, demonstrated by decreased phosphorylation of the mTORC1 targets S6K and 4EBP1.[1]
In a panel of lung cancer cell lines (H358, H661, H23, H460, A549) and K562 cells, treatment with SMI-4a (5 μM for 1 hour) stimulated AMPK phosphorylation only in LKB1-containing cells (H358, H661, K562), but not in LKB1-deficient cells (H23, H460, A549), indicating that SMI-4a requires LKB1 to activate AMPK.[1]

SMI-4a (60 mg/Kg) treatment twice daily significantly reduce tumor size and is well tolerated. Tumors harvested 1 hour after the final oral gavage of SMI-4a demonstrates decreased phosphorylation of p70 S6K compared with tumors from mice treated with vehicle, whereas in comparison total p70 S6K expression isunchanged.
In a subcutaneous xenograft model using 6812/2 murine pre-T-LBL cells in Nu/nu nude mice, oral administration of SMI-4a at 60 mg/kg twice daily (5 days per week for 21 days) significantly decelerated tumor growth compared to vehicle (P<0.05). Once-daily dosing did not achieve significant effect. [1]
Tumors harvested 1 hour after the final oral gavage showed decreased phospho-p70 S6K, increased phospho-ERK1/2, and reduced MYC protein levels (to 81.3±12.1% of control, P<0.05). These biochemical effects were absent 60 hours after the last dose. [1]

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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Coupled kinase assay for IC50 determination: Assays were carried out in 20 mmol/L MOPS (pH 7) containing 100 mmol/L NaCl, 10 mmol/L MgCl2, 2.5 mmol/L phosphoenolpyruvate, 0.2 mmol/L NADH, 30 μg/mL pyruvate kinase, 10 μg/mL lactate dehydrogenase, 2 mmol/L DTT, and 25 nmol/L Pim-1 with 100 μmol/L peptide substrate (RSRHSSYPAGT). Activity was measured by monitoring NADH oxidation as the decrease at 340 nm. Reactions were initiated by addition of ATP (100 μmol/L). IC50 values determined by nonlinear regression. [1]
In vitro phosphorylation of full-length protein substrates: Purified Pim-1 (1 ng) was added along with 4E-BP1 (2 μg) or p27Kip1 (2 μg) and inhibitor in assay buffer [20 mmol/L MOPS (pH 7), 100 mmol/L NaCl, 10 mmol/L MgCl2, 2 mmol/L DTT]. Assays were initiated by addition of ATP (100 μmol/L) and [γ-32P]ATP (10 μCi). Reactions proceeded for 15 min at 30°C, then separated by SDS-PAGE. 32P-phosphorylated substrates visualized by autoradiography and quantified by densitometry. [1]

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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In K562 human erythroleukemia cells, SMI-4a was used at 5 μM concentration for 1 hour in serum-free medium. Cells were treated with DMSO as control. Following treatment, lysates were prepared and analyzed by Western blotting for phosphorylated and total protein levels of AMPKα, ACC, raptor, S6K, and 4EBP1.[1]
For siRNA experiments, K562 cells were transfected with scrambled siRNA or Pim-1 siRNA using a lipofectamine-based reagent. After 48 hours, lysates were probed for AMPK phosphorylation.[1]

Dissolved in 65% DMSO, 30% PEG-400, 5% Tween-80; 75, 60 mg/kg; oral administration Nu/nu nude mice injected with pre-T-LBL cells

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For cell culture experiments, K562 cells were treated with SMI-4a at 5 μM for 1 hour in the absence of serum. In lung cancer cell line experiments, cells were treated with DMSO, SMI-4a (5 μM), or SMI-16a (another Pim inhibitor, 5 μM) for 1 hour in serum-free medium, and then lysates were prepared for Western blotting.[1]

Oral pharmacokinetics in WT-FVB mice: after 60 mg/kg gavage, plasma Cmax ~200 μM at 1 hour; t1/2 ≈ 6 hours; eliminated by 24 hours. No accumulation after twice-daily dosing for 5 days. [1]

No significant adverse effects observed in mice treated with SMI-4a at 60 mg/kg BID for 21 days: no body weight loss (supplemental Figure 3B); white blood cell counts, hemoglobin, platelet counts unchanged compared to vehicle; no morphologic abnormalities on blood smears. Blood chemistry: alanine transferase, albumin, total protein, alkaline phosphatase, amylase, total bilirubin normal, indicating no hepatotoxicity, renal, or pancreatic dysfunction. [1]
Note: The thiazolidinedione chemotype has been associated with hepatotoxicity in other studies (reference 37), but no such toxicity was observed here. [1]

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.

Serine/threonine Pim kinases are overexpressed in solid tumors and hematologic malignancies, promoting cell growth and survival. This study found that a novel Pim kinase inhibitor, SMI-4a or Pim-1 siRNA, can activate mTORC1 by stimulating phosphorylation of AMP-dependent protein kinase (AMPK), a negative regulator of mTORC1, thereby blocking the activity of rapamycin-sensitive mammalian target of rapamycin (mTORC1). Mouse embryonic fibroblasts (MEFs) with all three Pim kinases knocked out (triple knockout (TKO) MEFs) exhibited AMPK activation due to their higher AMP-ATP ratio compared to wild-type MEFs. Consistent with these findings, TKO MEFs grew slowly in vitro and exhibited reduced protein synthesis rates due to decreased 5' cap-dependent translation. Expression of Pim-3 alone in TKO MEF cells was sufficient to reverse AMPK activation, increase protein synthesis, and promote MEF cell growth, bringing them closer to wild-type. Studies have shown that Pim-3 expression significantly increases the protein levels of c-Myc and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), two enzymes that regulate glycolysis and mitochondrial biosynthesis, while their expression levels are reduced in TKO MEF cells. Overexpression of PGC-1α in TKO MEF cells increases ATP levels and inhibits AMPK activation. These results suggest that Pim kinase mediates the regulation of energy metabolism, thereby modulating AMPK activity. We found that Pim-3 plays an important role in regulating c-Myc and PGC-1α protein levels and cell growth. [1]

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SMI-4a is a benzylidene-thiazolidine-2,4-dione derivative that inhibits Pim kinase activity. Previous studies (referenced in this paper) have shown that SMI-4a induces apoptosis of human leukemic cells and synergizes with rapamycin to downregulate 4EBP1 phosphorylation and inhibit cell growth. In the current study, SMI-4a is used as a tool to demonstrate that Pim kinase inhibition activates AMPK in an LKB1-dependent manner, leading to decreased mTORC1 activity and cap-dependent translation. The compound is not evaluated in vivo in this paper.[1]

C11H6F3NO2S

273.23

273.007

C, 48.36; H, 2.21; F, 20.86; N, 5.13; O, 11.71; S, 11.73

327033-36-3

1361334

Typically exists as solid at room temperature

1.5±0.1 g/cm3

1.602

2.3

1

6

1

18

406

0

O=C(NC/1=O)SC1=C\C2=CC=CC(C(F)(F)F)=C2

NGJLOFCOEOHFKQ-VMPITWQZSA-N

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+

(5E)-5-[[3-(trifluoromethyl)phenyl]methylidene]-1,3-thiazolidine-2,4-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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