PIM1 (IC50 = 24 μM;(Ki = 0.6 μM); PIM2 (IC50 = 100 μM)

In intact cells, SMI-4a treatment (0.5 μM; 1 hour; HEK-293T cells) attenuates autophosphorylation of tagged Pim-1 [1].
The present study aimed to explore the mechanism underlying the antitumor effect of SMI‑4a in K562 and imatinib‑resistant K562 (K562/G) cell lines. It was demonstrated that SMI‑4a inhibited the proliferation of K562 and K562/G cells using a WST‑8 assay. The Annexin V‑propidium iodide assay demonstrated that SMI‑4a induced apoptosis of K562 and K562/G cells in a dose‑, and time‑dependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI‑4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI‑4a decreased phosphorylated (p)‑Ser9‑glycogen synthase kinase (GSK) 3β/pGSK3β and inhibited the translocation of β‑catenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADP‑ribose) polymerase‑1 was upregulated, and apoptosis regulator Bcl‑2 and Myc proto‑oncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of β‑catenin in the plasma and nucleus. The results of the present study suggest that SMI‑4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.Reference: Mol Med Rep. 2017 Oct;16(4):4603-4612. https://pubmed.ncbi.nlm.nih.gov/28849186/

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.

Pim Kinase Assays[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.

Western Blot Analysis[1]
Cell Types: HEK-293T cells
Tested Concentrations: 0.5 µM
Incubation Duration: 1 hour
Experimental Results: Caused a dose-dependent reduction in Pim-1-induced 4E-BP1 phosphorylation, with an IC50 of approximately 125 nM.

Antitumor Assay[1]
A syngeneic mouse tumor model that uses a transformed murine mammary adenocarcinoma cell line (JC, ATCC number CRL-2116) and Balb/C mice (Charles River) was performed as previously described. Tumor cells (1 × 106) were implanted subcutaneously, and tumor volume was calculated using the equation: (L × W2)/2. Upon detection of tumors, mice were randomized into treatment groups. Treatment was then administered once per day, five days per week, thereafter consisting of intraperitoneal doses of 0 or 50 mg 16a/kg or vehicle (50% DMSO: 50% phosphate-buffered saline). Whole body weights and tumor volume measurements were performed three times per week.

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Treatment protocols: Female (NZB × NZW)F1 mice were orally treated with AZD1208 (15 mg/kg) or vehicle control (0.1% Tween 80 and 0.5% methyl cellulose in water) 19, 20 for 12 weeks (n = 10 mice per group), starting at age 22 weeks (at the time of onset of proteinuria). Mice were then placed under anesthesia and killed at age 34 weeks. Twelve-week-old MRL/lpr mice received the selective Pim-1 inhibitor SMI-4a (60 mg/kg) or vehicle control (DMSO/PEG-400/Tween 80) twice daily, as described previously 21. Oral gavage was administered on 5 of 7 days each week for 8 weeks (n = 10 mice per group). In an independent experiment, survival was observed in mice until age 30 weeks, and the survival rates were compared between 2 groups (n = 15 mice per group). Reference: Arthritis Rheumatol. 2019 Aug;71(8):1308-1318. https://onlinelibrary.wiley.com/doi/abs/10.1002/art.40863

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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

[1]. Synthesis and evaluation of novel inhibitors of Pim-1 and Pim-2 protein kinases. J Med Chem. 2009 Jan 8;52(1):74-86.

The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (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 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. Computational docking of 4a to Pim-1 provided a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized. The most potent new compounds exhibited IC(50)s of 13 nM for Pim-1 and 2.3 microM for Pim-2. Additional compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim-2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall, these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.[1]

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The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus‑1 (PIM‑1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis.

C11H6F3NO2S

273.23

273.007

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

438190-29-5

1361334

White to off-white solid powder

1.5±0.1 g/cm3

1.602

2.3

1

6

1

18

406

0

S1C(N([H])C(/C/1=C(\[H])/C1C([H])=C([H])C([H])=C(C(F)(F)F)C=1[H])=O)=O

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+

5-(3-(trifluoromethyl)benzylidene)thiazolidine-2,4-dione

TCS PIM-1 4a; SMI-4a; TCS PIM-1 4a; (Z)-SMI-4a; (5Z)-5-[3-(trifluoromethyl)benzylidene]-1,3-thiazolidine-2,4-dione; (Z)-5-(3-(trifluoromethyl)benzylidene)thiazolidine-2,4-dione; CHEMBL183906; SMI4a; SMI 4a

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.)