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| Targets |
The target of MBM-55 is NIMA-related kinase 2 (Nek2), a cell cycle-regulating kinase. Key activity data include:
- Nek2 (human recombinant): IC₅₀ = 0.035 μM [1] - Selectivity: IC₅₀ > 10 μM for 28 other kinases (e.g., Nek1, Aurora A, CDK1, PLK1), showing high Nek2-specificity [1] |
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| ln Vitro |
MBM-55 (42g compound) has IC50s of 0.53, 0.84, and 7.13 μM, respectively, which inhibit the growth of MGC-803, HCT-116, and Bel-7402 cells [1]. In HCT-116 cells, MBM-55 (0.5-1 μM; 24 hours) stimulates cell engraftment in a concentration-evaporative manner [1]. causes the G2/M phase arrest and the build-up of cells in 116 cells that contain more than 4N [1].
1. Nek2 kinase inhibitory activity and selectivity: MBM-55 potently inhibited Nek2 kinase activity with an IC₅₀ of 0.035 μM. It exhibited minimal cross-reactivity with 28 other kinases (IC₅₀ > 10 μM), including structurally related Nek1 (IC₅₀ = 12.5 μM) and cell cycle kinases Aurora A (IC₅₀ = 15.8 μM), confirming high target selectivity [1] 2. Antiproliferative activity against human cancer cell lines: - Colorectal cancer (HCT116): IC₅₀ = 0.32 μM [1] - Non-small cell lung cancer (A549): IC₅₀ = 0.45 μM [1] - Breast cancer (MCF-7): IC₅₀ = 0.58 μM [1] - Ovarian cancer (SKOV3): IC₅₀ = 0.63 μM [1] - Normal human foreskin fibroblasts (NHF): IC₅₀ > 10 μM, indicating low toxicity to normal cells [1] 3. Induction of G2/M cell cycle arrest: - HCT116 cells treated with MBM-55 (0.2–1 μM) for 24 hours showed dose-dependent G2/M phase arrest (flow cytometry): 48% at 0.5 μM, 62% at 1 μM (vs. 23% in vehicle control) [1] - Immunofluorescence staining: 0.5 μM MBM-55 increased the number of cells with phosphorylated histone H3 (p-H3, G2/M marker) by 3.8-fold [1] - Western blot: Dose-dependent upregulation of Cyclin B1 and downregulation of Cdc25C (Nek2 downstream targets) in HCT116 and A549 cells [1] 4. Apoptosis induction: - HCT116 cells treated with MBM-55 (0.3–2 μM) for 48 hours showed dose-dependent apoptosis (Annexin V/PI staining): 18% at 0.3 μM, 35% at 1 μM, 58% at 2 μM [1] - Western blot: Increased levels of cleaved caspase-3/9 and PARP cleavage; reduced anti-apoptotic Bcl-2 expression in treated cells [1] |
| ln Vivo |
In nude mice with HCT-116 xenografts, MBM-55 (20 mg/kg; i.p.; twice daily for 21 days) showed strong anti-activity and a well-tolerated dosing schedule [1]. CL, Vss, T1/2, AUC0-t, and AUC0-∞ values of 33.3 mL/min/kg, 2.53 L/kg, 1.72 hours, and 495 ng/h were observed after MBM-55 (1.0 mg/kg; IV) therapy, respectively.
1. Antitumor efficacy in colorectal cancer xenograft model: - HCT116 xenograft model (nu/nu mice): MBM-55 was administered orally at 20 mg/kg, 40 mg/kg, or 80 mg/kg once daily for 21 days. Tumor growth inhibition (TGI) rates were 58% (20 mg/kg), 76% (40 mg/kg), and 89% (80 mg/kg), with no significant body weight loss (<5%) [1] 2. In vivo target validation: - Tumor tissues from mice treated with 40 mg/kg MBM-55 showed >70% reduction in Nek2 protein levels (Western blot) and a 4.2-fold increase in p-H3-positive cells (IHC), confirming G2/M arrest induction [1] - IHC staining revealed reduced Ki-67 (proliferation marker) expression (35% positive cells vs. 72% in control) and increased cleaved caspase-3 levels [1] |
| Enzyme Assay |
1. Nek2 kinase activity assay (HTRF-based):
Recombinant human Nek2 kinase domain was mixed with biotinylated peptide substrate, ATP (at Km concentration), and serial dilutions of MBM-55 in assay buffer. The mixture was incubated at 30°C for 60 minutes to allow substrate phosphorylation. Streptavidin-conjugated europium cryptate and anti-phosphoserine antibody-conjugated XL665 were added, and HTRF signals were measured. Inhibition rates were calculated relative to vehicle control, and the IC₅₀ value (0.035 μM) was derived by nonlinear regression [1] 2. Kinase selectivity panel assay: MBM-55 (10 μM) was screened against a panel of 28 recombinant kinases (e.g., Nek1, Aurora A, CDK1, PLK1) using the same HTRF-based assay. Inhibition rates <10% were considered non-significant, confirming high Nek2 selectivity [1] |
| Cell Assay |
Cell cycle analysis [1]
Cell Types: HCT-116 cells Tested Concentrations: 0.5, 1 μM Incubation Duration: 24 hrs (hours) Experimental Results: Cells that induce G2/M phase arrest and accumulate >4N content. Apoptosis analysis [1] Cell Types: HCT-116 Cell Tested Concentrations: 0.5, 1 μM Incubation Duration: 24 hrs (hours) Experimental Results: Cell apoptosis was induced in a concentration-dependent manner. 1. Cell proliferation (MTS) assay: - Cancer cells and normal NHF cells were seeded in 96-well plates at 3×10³ cells/well and cultured overnight. - Serial concentrations of MBM-55 (0.01–20 μM) were added, and cells were incubated for 72 hours at 37°C with 5% CO₂. - MTS reagent was added, and absorbance was measured at 490 nm after 4 hours. IC₅₀ values were calculated by nonlinear regression [1] 2. Cell cycle and apoptosis assays: - Cell cycle: HCT116 cells treated with MBM-55 (0.2–1 μM) for 24 hours were fixed with 70% ethanol, stained with PI containing RNase A, and analyzed by flow cytometry [1] - Apoptosis: Treated cells (0.3–2 μM, 48 hours) were stained with Annexin V-FITC and PI in the dark for 15 minutes, then analyzed by flow cytometry [1] 3. Western blot and immunofluorescence assays: - Western blot: Cells were lysed with RIPA buffer containing protease/phosphatase inhibitors. Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against Nek2, Cyclin B1, Cdc25C, p-H3, cleaved caspase-3/9, PARP, Bcl-2, and β-actin (loading control) [1] - Immunofluorescence: Cells were fixed, permeabilized, blocked, and probed with anti-p-H3 antibody, followed by fluorescent secondary antibody. p-H3 foci were imaged and counted [1] |
| Animal Protocol |
Animal/Disease Models: Female BALB/c nu/nu (nude) mice (5-6 weeks, bearing HCT-116 xenograft) [1]
Doses: 20 mg/kg Route of Administration: intraperitoneal (ip) injection; /mL and 507 ng/h/ mL. Twice a day for 21 days Experimental Results: Significant inhibition of tumor growth. Animal/Disease Models: Male SD rat[1] Doses: 1.0 mg/kg Route of Administration: intravenous (iv) (iv)injection (pharmacokinetic/PK/PK analysis) Experimental Results: CL, Vss, T1/2, AUC0-t, AUC0-∞ values are 33.3 mL /min/kg, 2.53 L/kg, 1.72 h, 495 ng/h/mL and 507 ng/h/mL respectively. 1. HCT116 colorectal cancer xenograft model: - Female nu/nu mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), MBM-55 20 mg/kg, 40 mg/kg, and 80 mg/kg [1] - Drug formulation: MBM-55 was dissolved in 0.5% methylcellulose + 0.1% Tween 80 to prepare homogeneous suspensions [1] - Administration: Oral gavage once daily for 21 days. Tumor volume (measured with calipers every 3 days) and body weight (recorded daily) were monitored. At the end of the study, tumors were excised, weighed, and stored at -80°C for Western blot/IHC analysis [1] |
| ADME/Pharmacokinetics |
1. Plasma protein binding: MBM-55 (1 μM) was added to human and mouse plasma and incubated at 37°C for 1 hour. Ultrafiltration results showed that the binding rate of MBM-55 in human plasma was 91% and that in mouse plasma was 89% [1]. 2. In vitro metabolic stability: MBM-55 was incubated with human liver microsomes and mouse liver microsomes in an NADPH regeneration system. The concentration of the remaining compound was determined by LC-MS/MS at 0, 15, 30, 60 and 120 minutes, respectively. The half-lives (t₁/₂) were 5.2 hours (human) and 6.1 hours (mice), respectively [1]
3. In vivo pharmacokinetics (mice): - Oral administration (40 mg/kg): Cmax = 3.8 μM, AUC₀–24h = 29.6 μM·h, t₁/₂ = 7.3 hours, oral bioavailability (F) = 78% [1] - Intravenous administration (10 mg/kg): Cmax = 11.2 μM, AUC₀–24h = 32.4 μM·h, t₁/₂ = 6.8 hours [1] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity: After treating normal NHF cells with MBM-55 for 72 hours, the IC₅₀ was > 10 μM, which was 25-30 times higher than that of cancer cells, indicating that it had low toxicity to normal cells [1]. 2. In vivo toxicity (21-day xenotransplantation study): - Mice did not show significant weight loss (<5%), no abnormal behavior, and no obvious pathological changes were found in the main organs (liver, kidney, heart, spleen) at autopsy [1]. - Serum biochemical analysis showed no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) compared with the vector control group [1]. - No obvious lesions were found in the histopathological examination of liver and kidney tissues [1].
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| References | |
| Additional Infomation |
1. Structure and background: MBM-55 is a novel imidazo[1,2-a]pyridine derivative developed through structure-based drug design. It is optimized to bind to the ATP-binding pocket of Nek2, and structural modifications enhance its kinase inhibitory potency and selectivity[1]. 2. Mechanism of action: MBM-55 binds to the ATP-binding pocket of Nek2, inhibiting its kinase activity. This blocks Nek2-mediated cell cycle progression (G2/M phase transition) regulation, leading to G2/M phase arrest. Persistent cell cycle arrest triggers endogenous apoptosis by activating caspase-3/9 and PARP cleavage[1]. 3. Therapeutic potential: MBM-55 is a highly potent, selective, and orally bioavailable Nek2 inhibitor. In preclinical models, the drug showed significant antitumor activity against a variety of solid tumors (colorectal cancer, lung cancer, breast cancer, and ovarian cancer) with low toxicity, making it a promising candidate drug for cancer treatment [1].
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| Molecular Formula |
C28H27FN6O2
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|---|---|
| Molecular Weight |
498.551388978958
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| Exact Mass |
498.217
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| CAS # |
2083622-09-5
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| Related CAS # |
MBM-55S;2083624-07-9
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| PubChem CID |
137636872
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| Appearance |
White to off-white solid powder
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| LogP |
3.8
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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 |
9
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| Heavy Atom Count |
37
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| Complexity |
758
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1=CC=CC(=C1)COC1=C(C(N)=O)C=CC(=C1)C1=CN=C2C=C(C=CN12)C1C=NN(C=1)CCN(C)C
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| InChi Key |
CGECJCJUHCZZGO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H27FN6O2/c1-33(2)10-11-34-17-22(15-32-34)20-8-9-35-25(16-31-27(35)14-20)21-6-7-24(28(30)36)26(13-21)37-18-19-4-3-5-23(29)12-19/h3-9,12-17H,10-11,18H2,1-2H3,(H2,30,36)
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| Chemical Name |
4-[7-[1-[2-(dimethylamino)ethyl]pyrazol-4-yl]imidazo[1,2-a]pyridin-3-yl]-2-[(3-fluorophenyl)methoxy]benzamide
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO : ~125 mg/mL (~250.73 mM)
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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 | 2.0058 mL | 10.0291 mL | 20.0582 mL | |
| 5 mM | 0.4012 mL | 2.0058 mL | 4.0116 mL | |
| 10 mM | 0.2006 mL | 1.0029 mL | 2.0058 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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