CerK (IC50 = 12 nM); apoptosis
Ceramide kinase (CerK) (Ki = 7.4 nM for ceramide binding; in vitro IC50 = 12 ± 2 nM; cellular IC50 = 59.7 ± 12 nM in MCF-7 cells overexpressing CerK) [1][2]
Diacylglycerol kinase α (DAGKα) (IC50 = 5 μM, approximately 500-fold less potent than CerK) [1]
Sphingosine kinase 1 (SphK1) (IC50 > 100 μM, >10,000-fold selectivity) [1]
Sphingosine kinase 2 (SphK2) (IC50 > 100 μM, >10,000-fold selectivity) [1]
Phosphoinositide 3-kinase α (PI3Kα), phosphatidylinositol 4-kinase β (PI4Kβ), Vps34, glucosylceramide synthase (GCS), sphingomyelin synthase 1 (SMS-1), ceramide transfer protein (CERT) (all IC50 >10-25 μM, >1000-fold selectivity) [1]
NVP-231 targets ceramide kinase (CerK), an enzyme that phosphorylates ceramide to produce ceramide-1-phosphate (C1P). C1P is a bioactive lipid involved in cell proliferation, survival, and inflammation. By competitively inhibiting the binding of ceramide to CerK, NVP-231 prevents the formation of C1P. This leads to decreased endogenous C1P levels.

NVP-231 causes cancer cells to die, which lowers cell viability and DNA synthesis. Cells treated with NVP-231 exhibited M phase arrest as opposed to G2/M boundary arrest. NVP-231 treatment results in a concentration-dependent up-regulation of cyclin B1 phosphorylation at Ser133 and a decrease of CDK1 phosphorylation at Tyr15 in MCF-7 and NCI-H358 cells. NVP-231 inhibits Wee1 expression in NCI-H358 and MCF-7 cells. Cancer cells treated with NVP-231 exhibit concentration- and time-dependent down-regulation of the CDK4 protein. In synchronized cells, this effect is even more pronounced[2].

---

NVP-231 inhibited recombinant human CerK in vitro with an IC50 of 12 ± 2 nM and a Ki of 7.4 nM (competitive with ceramide, non-competitive with ATP). Inhibition was instantaneous and fully reversible. [1]
In cell-based assays using COS-1 cells overexpressing human CerK (COS-CerK) and fluorescent NBD-ceramide, NVP-231 inhibited NBD-C1P formation with an IC50 of approximately 60 nM and achieved complete inhibition at 100 nM. It did not inhibit glucosylceramide (GlcCer) or sphingomyelin (SM) formation; instead it increased those metabolites. [1]
In mouse peritoneal macrophages and bone marrow-derived mast cells, NVP-231 (50-100 nM) reduced C1P formation to background levels similar to CerK(-/-) cells. [1]
NVP-231 (100 nM) prevented phosphorylation of endogenous ceramides in mouse bone marrow-derived macrophages (BMDMs) as shown by 33P-orthophosphate labeling and 2D-TLC, whereas the inactive analog NVP-995 had no effect. LC/MS analysis showed that NVP-231 (100 nM) rapidly reduced C16-C1P levels to <20% of control in COS-CerK cells. [1]
In MCF-7 breast cancer and NCI-H358 lung cancer cells, NVP-231 concentration-dependently reduced cell viability (IC50 ~1 μM for MCF-7, ~500 nM for NCI-H358), DNA synthesis (BrdU incorporation), and colony formation (complete inhibition at 500 nM-1 μM). [2]
NVP-231 (1 μM) induced cell death as measured by propidium iodide uptake (20% in MCF-7, >40% in NCI-H358 at 72 h) and increased cleaved caspase-3 and caspase-9 (apoptosis). [2]
Flow cytometry analysis showed that NVP-231 (24 h) decreased S-phase cells and increased G2/M population (4N DNA content) in both cell lines. Phospho-histone H3 (Ser10) staining revealed that cells arrested in M phase (mitotic index increased up to 3-4 fold). Western blot showed increased cyclin B1 phosphorylation (Ser133), decreased CDK1 Tyr15 phosphorylation, decreased wee1 expression, and decreased CDK4 expression. [2]
Combination of NVP-231 (100 nM) with staurosporine (20 nM) synergistically increased DNA fragmentation and G2/M arrest. siRNA knockdown of CerK sensitized cells to staurosporine-induced apoptosis, while CerK overexpression protected cells. [2]
NVP-231 had no effect on PLA2G4A-dependent arachidonic acid or PGE2 release in neutrophils, macrophages, or fibroblasts. It did not accelerate spontaneous apoptosis in human blood neutrophils nor affect macrophage survival in the presence or absence of M-CSF. [1]
In vitro, NVP-231 is a potent CerK inhibitor with an IC₅₀ of 12 nM. At 10 nM, it inhibits ceramide-1-phosphate formation by >50%, and at 100 nM, it completely inhibits its formation in cell culture. It is active in the low nanomolar range on purified as well as cellular CerK. It induces cell apoptosis by increasing DNA fragmentation and caspase-3 and caspase-9 cleavage.

In vivo, NVP-231 has the potential to modulate C1P-mediated signaling pathways. By inhibiting CerK, it may have effects on cell proliferation, survival, and inflammation. However, specific in vivo efficacy data in animal models are not extensively detailed in the available literature. It is a research compound and is not approved for clinical use.

High-throughput screening assay: A homogeneous luminescent assay measuring ATP consumption (Kinase-Glo) was miniaturized to 1536-well format. Recombinant full-length human CerK (16 ng/μl) was incubated with 1 mM ceramide-containing micelles, 10 μM ATP, and test compounds (100 nl of 0.5 mM intermediate dilution) in reaction buffer (25 mM MOPS pH 7.2, 6.25 mM CaCl2, 62.5 mM KCl, 2.5 mM EGTA, 1.25 mM DTT) for 90-120 min at 37°C, followed by addition of Kinase-Glo reagent and luminescence measurement. Z' factor ~0.5. Hits were validated by 8-point dilution series and IC50 determination. [1]
In vitro radioactive CerK assay: 40 ng/ml GST-CerK was incubated with 180 μM C8 ceramide and 500 μM 32P-ATP in reaction buffer. Reactions were stopped, lipids extracted, and C1P quantified. IC50 of NVP-231 determined as 12±2 nM. [1]
Mechanism study: ATP competition (various ATP concentrations with 1 μM NVP-231) showed non-competitive inhibition toward ATP; ceramide competition (various ceramide concentrations with 0.1 μM NVP-231) showed competitive inhibition toward ceramide. Ki = 7.4 nM. [1]
Reversibility assay: CerK was pre-incubated with NVP-231, then diluted 100-fold; activity recovered fully, indicating reversible inhibition. [1]
In vitro CerK enzyme assays for NVP-231 involve measuring the phosphorylation of ceramide to C1P by ceramide kinase. The enzyme is incubated with ceramide, ATP, and varying concentrations of NVP-231. The production of C1P is measured using radiometric (³P-ATP) or mass spectrometry-based methods. IC₅₀ values are calculated from dose-response curves.

MCF-7 and NCI-H358 cells are plated in a 96-well black plate at a density of 1 × 104 cells per well, and they are then exposed to the indicated concentrations of NVP-231 (in nM) for 48 hours. During the final 4 hours of the treatment, alamarBlueR is added, and fluorescence is measured.

---

Cell-based CerK activity assay (NBD-ceramide): Cells were incubated with 5 μM NBD-C6-ceramide for 2 h in the presence of NVP-231. Lipids were extracted, separated by TLC, and NBD-C1P, NBD-GlcCer, NBD-SM quantified by fluorescence imaging. [1][2]
Cell viability assay (AlamarBlue): Cells in 96-well plates were treated with NVP-231 for 48 h, then resazurin added for 4 h, fluorescence measured at 544/590 nm. [2]
DNA synthesis (BrdU ELISA): Cells treated with NVP-231 for 72 h, BrdU added for last 24 h, incorporated BrdU detected by ELISA. [2]
Colony formation assay: Cells seeded at 1000 cells/dish, treated with NVP-231 for 10-14 days, colonies stained with crystal violet and counted. [2]
Cell death (propidium iodide uptake): Treated cells trypsinized, stained with PI (10 μg/ml), analyzed by flow cytometry for PI-positive cells. [2]
Apoptosis (caspase cleavage): Cell lysates separated by SDS-PAGE, Western blot with anti-cleaved caspase-3 and caspase-9 antibodies. [2]
Cell cycle analysis: Cells fixed in 70% ethanol, stained with PI (10 μg/ml) and RNase A (100 μg/ml), analyzed by flow cytometry for DNA content. Mitotic index determined by anti-phospho-histone H3 (Ser10) staining (Alexa-488 secondary). [2]
Western blot: Antibodies against phospho-Ser133 cyclin B1, total cyclin B1, phospho-Tyr15 CDK1, total CDK1, wee1, CDK4, GAPDH, β-actin. [2]
Lipid analysis by LC-MS/MS: C16-C1P and ceramide subspecies quantified using C12-C1P and C17-ceramide as internal standards, on a Q-TOF 6530 mass spectrometer in positive (ceramides) or negative (C1P) ESI mode. [2]
In vitro cell-based assays for NVP-231 are performed using various cell lines. Cells are cultured in appropriate media and treated with NVP-231 at various concentrations. C1P levels in cell lysates are measured by LC-MS or ELISA. Cell viability is measured by MTT or CellTiter-Glo assays. Apoptosis is evaluated by caspase-3/7 activation and DNA fragmentation assays.

In vivo animal studies for NVP-231 would typically involve mouse models of cancer or inflammation. The compound is administered intraperitoneally or orally. Tumor growth or inflammatory responses are assessed. Pharmacodynamic studies measure C1P levels in tissues to confirm CerK inhibition.

Specific pharmacokinetic properties of NVP-231, such as half-life and oral bioavailability, are not extensively detailed in the available literature. As a small molecule with a molecular weight of approximately 431.55 g/mol, it is expected to have moderate oral bioavailability. It is soluble in DMSO and is typically formulated for in vivo administration.

Comprehensive toxicological data for NVP-231 are not widely available in public literature. As a research compound, it is intended for laboratory use only and is not for human therapeutic use. Standard safety precautions should be followed when handling this compound. The compound is supplied with a purity of ≥98%.

[1] Mol Pharmacol . 2008 Oct;74(4):925-32.

[2] Br J Pharmacol . 2014 Dec;171(24):5829-44.

NVP-231 belongs to the benzothiazole class of compounds.

---

NVP-231 is a first-in-class potent and specific ceramide kinase inhibitor. It was discovered by high-throughput screening of over one million compounds. The compound is cell-permeable, reversible, and competes with ceramide binding. It serves as a valuable tool to study CerK biology. In cancer cells, CerK inhibition leads to M phase arrest, reduced CDK4 expression, and apoptosis. The compound shows synergy with staurosporine. CerK overexpression protects cells from apoptosis. NVP-231 has been used to demonstrate that CerK is involved in cell cycle progression, particularly M phase regulation. [1][2]
NVP-231 is a potent, specific, and reversible inhibitor of ceramide kinase (CerK). It has an IC₅₀ of 12 nM. It competitively inhibits ceramide binding to CerK and decreases endogenous C1P levels. It induces apoptosis. This product is for research use only.

C25H25N3O2S

431.5499

431.166

C, 69.58; H, 5.84; N, 9.74; O, 7.41; S, 7.43

362003-83-6

362003-83-6

4096211

White to off-white solid powder

1.4±0.1 g/cm3

1.743

5.34

2

4

4

31

680

0

S1C(N([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=O)=NC2C([H])=C([H])C(=C([H])C1=2)N([H])C(C12C([H])([H])C3([H])C([H])([H])C([H])(C([H])([H])C([H])(C3([H])[H])C1([H])[H])C2([H])[H])=O

MVSSJPGNLQPWSW-UHFFFAOYSA-N

InChI=1S/C25H25N3O2S/c29-22(18-4-2-1-3-5-18)28-24-27-20-7-6-19(11-21(20)31-24)26-23(30)25-12-15-8-16(13-25)10-17(9-15)14-25/h1-7,11,15-17H,8-10,12-14H2,(H,26,30)(H,27,28,29)

N-(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide

NVP231; NVP 231; NVP-231

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)

DMSO: ≥41 mg/mL (~95.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.79 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.79 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.79 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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