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K145 (K-145) is a novel, potent and selective SphK2 (sphingosine kinase-2) inhibitor with anticancer effects. It inhibits SphK2 with an IC50 of 4.30±0.06 μM and exhibits no inhibition against SphK1 at concentrations up to 10 μM. As a selective sphingosine kinase-2 (SphK2) inhibitor, K145 has anticancer activity and inhibited the activity of SphK2 in a dose-dependent manner. Biochemical assay results indicate that K145 is a selective SphK2 inhibitor. Molecular modeling studies also support this notion. In vitro studies using human leukemia U937 cells demonstrated that K145 accumulates in U937 cells, suppresses the S1P level, and inhibits SphK2. K145 also exhibited inhibitory effects on the growth of U937 cells as well as apoptotic effects in U937 cells, and that these effects may be through the inhibition of down-stream ERK and Akt signaling pathways. K145 also significantly inhibited the growth of U937 tumors in nude mice by both intraperitoneal and oral administration, thus demonstrating its in vivo efficacy as a potential lead anticancer agent. The antitumor activity of K145 was also confirmed in a syngeneic mouse model by implanting murine breast cancer JC cells in BALB/c mice. Collectively, these results strongly encourage further optimization of K145 as a novel lead compound for development of more potent and selective SphK2 inhibitors.
| Targets |
K145 is a selective sphingosine kinase-2 (SphK2) inhibitor (IC₅₀ = 4.30 ± 0.06 µM). It competitively inhibits SphK2 with a Ki of 6.4 ± 0.7 µM (with respect to sphingosine). [1]
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| ln Vitro |
K145 (0-10 µM; 24-72 hours; U937 cells) therapy greatly reduces the proliferation of U937 cells in a concentration-dependent manner [1]. K145 (10 µM; 24 hours; U937 cells) treatment effectively promoted apoptosis in U937 cells [1]. K145 (4-8 µM; 3 hours; U937 cells) treatment lowers ERK and Akt phosphorylation [1]. K145 (10 µM) therapy resulted in a decrease in total cellular S1P but had no significant effect on ceramide levels [1].
K145 selectively inhibited recombinant SphK2 activity in a dose-dependent manner with an IC₅₀ of 4.30 ± 0.06 µM, while showing no inhibition of SphK1 at concentrations up to 10 µM. K145 (10 µM) decreased total cellular sphingosine-1-phosphate (S1P) levels in U937 cells without significantly affecting ceramide or ceramide-1-phosphate (C1P) levels. K145 (10 µM) inhibited the phosphorylation of FTY720, a SphK2-specific substrate, in U937 cells. K145 significantly inhibited the proliferation of human leukemia U937 cells in a concentration-dependent manner when cultured in 10% serum. K145 (10 µM, 24 h) induced apoptosis in U937 cells, primarily late apoptosis, with minimal necrosis. K145 treatment (as low as 4 µM, 1 h) inhibited the phosphorylation of ERK and Akt in U937 cells, indicating suppression of downstream survival signaling pathways. K145 showed relative selectivity against a panel of eleven other protein kinases at 10 µM concentration. [1] |
| ln Vivo |
The treatment of BALB/c-nu mice with K145 (50 mg/kg; oral gavage; daily; for 15 days) dramatically slowed the formation of U937 tumors in nude mice [1].
In a U937 xenograft model in nude mice, daily intraperitoneal (i.p.) administration of K145 (15 mg/kg for 17 days) significantly inhibited tumor growth, resulting in a tumor growth inhibition (TGI) of 44.2%. No significant body weight loss was observed. In a syngeneic JC mammary adenocarcinoma model in immunocompetent BALB/c mice, daily i.p. administration of K145 (20 mg/kg and 35 mg/kg for 15 days) significantly inhibited tumor growth in a dose-dependent manner. Tumor S1P levels were reduced, and phospho-ERK and phospho-Akt levels were decreased in tumor samples. No significant changes in body weight or major organ toxicity were observed. In a U937 xenograft model in nude mice, daily oral administration of K145 (50 mg/kg for 15 days) significantly inhibited tumor growth (TGI = 51.25%), demonstrating oral bioavailability and in vivo efficacy. No apparent toxicity was observed. [1] |
| Enzyme Assay |
Recombinant SphK1 and SphK2 activities were measured using cell lysates overexpressing the respective enzymes. SphK1 activity was assayed with 5 µM sphingosine and [γ-³²P]ATP in the presence of 0.25% Triton X-100 to inhibit SphK2. SphK2 activity was assayed with sphingosine added as a complex with BSA and [γ-³²P]ATP in the presence of 1 M KCl to inhibit SphK1. Inhibition was measured in the presence of varying concentrations of K145 or control inhibitors.
Ceramide kinase (CERK) activity was assayed using a luminescent kinase assay. Recombinant CERK was incubated with ceramide and ATP in the presence or absence of K145. Luminescence was measured after adding a detection reagent. [1] |
| Cell Assay |
Cell viability assay [1]
Cell Types: U937 cells Tested Concentrations: 0 µM, 4 µM, 6 µM, 8 µM, 10 µM Incubation Duration: 24 hrs (hours), 48 hrs (hours), 72 hrs (hours) Experimental Results: Dramatically inhibited the growth of U937 cells at a certain concentration Growth-dependent manner. Apoptosis analysis [1] Cell Types: U937 Cell Tested Concentrations: 10 µM Incubation Duration: 24 hrs (hours) Experimental Results: Dramatically induced apoptosis in U937 cells. Western Blot Analysis[1] Cell Types: U937 Cell Tested Concentrations: 4 µM, 8 µM Incubation Duration: 3 hrs (hours) Experimental Results: diminished phosphorylated ERK and Akt. Cell proliferation was assessed using the MTT assay. U937 cells were plated in 96-well plates, treated with K145 for 72 hours, then incubated with MTT reagent. The formazan product was dissolved in DMSO, and absorbance was measured at 570 nm. Apoptosis was analyzed by flow cytometry using Annexin V-FITC and propidium iodide (PI) double staining. U937 cells were treated with K145, harvested, washed, and stained according to the manufacturer's instructions. Cells were analyzed to distinguish early apoptotic (Annexin V+/PI-), late apoptotic (Annexin V+/PI+), and necrotic (Annexin V-/PI+) populations. Western blotting was performed to analyze signaling proteins. U937 cells were treated with K145, lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies against phospho-ERK, total ERK, phospho-Akt, total Akt, and GAPDH as a loading control. Cellular lipid levels (S1P, ceramide, C1P) and drug accumulation were measured by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Cells were treated with K145, harvested, washed, and lipids were extracted for analysis. [1] |
| Animal Protocol |
Animal/Disease Models: BALB/c-nu (nude) mice injected with U937 cells [1]
Doses: 50 mg/kg Route of Administration: po (oral gavage); daily; 15 days Experimental Results: po (oral gavage); daily; 50 mg/kg dose It inhibited U937 tumor growth for 15 days and no obvious toxicity was observed. For the U937 xenograft study in nude mice (BALB/c-nu), U937 cells were implanted subcutaneously. When tumors reached a palpable size, mice were treated daily with K145 (15 mg/kg) or vehicle via intraperitoneal injection for 17 days. Tumor volume and body weight were monitored regularly. For the syngeneic JC tumor model in BALB/c mice, JC mammary adenocarcinoma cells were implanted subcutaneously. Mice were treated daily with K145 (20 mg/kg or 35 mg/kg) or vehicle via intraperitoneal injection for 15 days. Tumor volume and body weight were monitored. For the oral efficacy study in the U937 xenograft model, nude mice bearing U937 tumors were treated daily with K145 (50 mg/kg) or vehicle via oral gavage for 15 days. Tumor volume and body weight were monitored. [1] |
| ADME/Pharmacokinetics |
K145 was absorbed by U937 cells in a concentration-dependent manner. K145 was detected in tumor tissues of mice treated with this compound. Treatment with K145 resulted in a decrease in S1P levels in tumor tissues. [1]
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| Toxicity/Toxicokinetics |
In the U937 xenograft model (intraperitoneal injection, 15 mg/kg), no decrease in body weight was observed in mice treated with K145 compared to the vector control group. In the homologous JC tumor model (intraperitoneal injection, 20 and 35 mg/kg), no significant changes in body weight or obvious toxicity to major organs (heart, lung, liver, kidney) were observed. In the oral U937 xenograft model (50 mg/kg), a slight decrease in initial body weight was observed, but the weight recovered during treatment, and no other obvious toxicity was observed. [1]
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| References | |
| Additional Infomation |
K145 (3-(2-aminoethyl)-5-[3-(4-butoxyphenyl)propylidene]-thiazolidin-2,4-dione) is a thiazolidin-2,4-dione (TZD) analog designed as a selective SphK2 inhibitor. Molecular modeling and docking studies have shown that K145 preferentially binds to the sphosinic-binding pocket of SphK2 rather than SphK1, possibly due to the difference in key interacting residues: SphK2 is glutamine (Gln), while SphK1 is glutamate (Glu). The antitumor effect of K145 is attributed to its inhibition of SphK2, leading to a decrease in S1P levels, which in turn inhibits downstream ERK and Akt survival signaling pathways, ultimately inducing apoptosis. K145 is considered a novel lead compound for the development of more effective and selective SphK2 inhibitors and potential anticancer drugs. [1]
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| Molecular Formula |
C₁₈H₂₄N₂O₃S
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|---|---|
| Molecular Weight |
348.46
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| Exact Mass |
348.151
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| CAS # |
1309444-75-4
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| Related CAS # |
K145 hydrochloride;1449240-68-9
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| PubChem CID |
71714682
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
3.974
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
24
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| Complexity |
459
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(N(CCN)C/1=O)SC1=C/CCC2=CC=C(OCCCC)C=C2
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| InChi Key |
MPZXLTZVPUSTFY-SOFYXZRVSA-N
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| InChi Code |
InChI=1S/C18H24N2O3S/c1-2-3-13-23-15-9-7-14(8-10-15)5-4-6-16-17(21)20(12-11-19)18(22)24-16/h6-10H,2-5,11-13,19H2,1H3/b16-6-
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| Chemical Name |
(5Z)-3-(2-aminoethyl)-5-[3-(4-butoxyphenyl)propylidene]-1,3-thiazolidine-2,4-dione
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| Synonyms |
K145 K-145 K 145
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 2.8698 mL | 14.3488 mL | 28.6977 mL | |
| 5 mM | 0.5740 mL | 2.8698 mL | 5.7395 mL | |
| 10 mM | 0.2870 mL | 1.4349 mL | 2.8698 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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