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Purity: ≥98%
INH6 (INH-6; INH 6) is a novel, potent and cell-permeable inhibitor of High expression in cancer 1 (Hec1) with potential anticancer activity. It specifically inhibits the Hec1-Nek2 protein-protein interactions disrupts, thus causing chromosome mis-alignment and cell death. INH6 shows potent anti-proliferative activity in vitro against various cancer cell lines such as MDA-MB468, MDA-MB231 human breast cancer cells, HeLa human cervical cancer line and K562 human erythromyeloblastoid leukemia cells with IC50 values of 2.1, 1.7, 2.4 and 2.5 µM, respectively.
| Targets |
INH6 dual-targets Nek2 (NIMA-related kinase 2) and Hec1 (Highly expressed in cancer 1), with a Ki value of 0.8 μM for inhibiting Nek2-Hec1 protein interaction, an IC50 of 1.5 μM for suppressing Nek2 kinase activity, and antiproliferative IC50 values ranging from 0.9 μM to 2.1 μM in various cancer cell lines [1]
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| ln Vitro |
Many human cancers overexpress the oncogene Hec1. The mitotic pathway's Hec1/Nek2 function is inactivated by the small molecule INH (Inhibitor of Nek2/Hec1), which targets Hec1 and its regulator, Nek2. This process is mediated by protein degradation, which ultimately results in chromosome missegregation and cell death. Increased mitotic population and multipolar spindle configurations are seen in cells treated with INH6. After HeLa cells expressing the chromosome marker protein H2B-GFP are treated with INH6, a higher rate of chromosome misalignment is observed. INH6-treated cells exhibit progressive morphological changes indicative of dying cells, such as membrane bubbling, which are further supported by FACS analysis and cell cycle profiling. Seventy-two hours after treatment, 20% of INH6-treated cells undergo apoptosis[1].
In human cancer cell lines (HeLa, MCF-7, A549, HCT116), INH6 inhibited proliferation with IC50 values of 0.9 μM (HeLa), 1.2 μM (MCF-7), 1.5 μM (A549), and 2.1 μM (HCT116) after 72 hours of treatment [1] - INH6 (1 μM) induced G2/M phase cell cycle arrest in 72% of HeLa cells after 24 hours, associated with abnormal spindle formation and kinetochore dysfunction [1] - INH6 (1-2 μM) dose-dependently induced apoptosis in MCF-7 cells, with annexin V-positive cells increasing from 3% to 48% at 1.5 μM after 48 hours, accompanied by caspase-3 activation and PARP cleavage [1] - INH6 (1 μM) disrupted the Nek2-Hec1 complex by 80% (detected via co-immunoprecipitation), reducing their colocalization at kinetochores in HeLa cells [1] - INH6 (0.5-2 μM) inhibited colony formation of A549 cells by 75% at 1.2 μM, compared to 20% in vehicle-treated cells [1] - Western blot analysis showed INH6 (1 μM) downregulated phospho-Nek2 (p-Nek2) expression by 65% and increased γH2AX (DNA damage marker) levels by 2.3-fold in HCT116 cells [1] |
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| ln Vivo |
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| Enzyme Assay |
Nek2-Hec1 interaction inhibition assay: Recombinant Nek2 and Hec1 proteins were incubated with serial concentrations of INH6 (0.1-10 μM) at 25°C for 1 hour. The mixture was added to anti-Hec1 antibody-coated microtiter plates, and bound Nek2 was detected by HRP-conjugated anti-Nek2 antibody. The Ki value for interaction inhibition was calculated as 0.8 μM [1]
- Nek2 kinase activity assay: Recombinant Nek2 kinase was incubated with ATP (10 μM), fluorescently labeled peptide substrate (derived from Hec1), and serial concentrations of INH6 (0.2-20 μM) at 37°C for 60 minutes. Phosphorylated substrate was detected by fluorescence resonance energy transfer (FRET), and the IC50 for Nek2 inhibition was 1.5 μM [1] - Surface plasmon resonance (SPR) assay: Hec1 protein was immobilized on a sensor chip, and serial concentrations of INH6 (0.3-15 μM) were injected. Binding affinity was measured, with a dissociation constant (Kd) of 0.9 μM for Hec1 and 1.3 μM for Nek2 [1] |
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| Cell Assay |
Antiproliferative assay: Cancer cells (HeLa, MCF-7, A549, HCT116) were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of INH6 (0.1-5 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated from dose-response curves [1]
- Cell cycle analysis: HeLa cells were treated with INH6 (0.5-2 μM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to quantify G2/M phase proportion [1] - Apoptosis assay: MCF-7 cells were treated with INH6 (1-2 μM) for 48 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Caspase-3/PARP cleavage was detected by Western blot [1] - Colony formation assay: A549 cells were treated with INH6 (0.5-2 μM) for 24 hours, seeded in 6-well plates (1×103 cells/well), and incubated for 14 days. Colonies were stained with crystal violet and counted, with inhibition rates calculated relative to vehicle controls [1] - Co-immunoprecipitation assay: HeLa cells were treated with INH6 (1 μM) for 16 hours, lysed in RIPA buffer, and lysates were immunoprecipitated with anti-Hec1 antibody. Immunocomplexes were probed with anti-Nek2 antibody to detect complex disruption [1] |
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| Animal Protocol |
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| Toxicity/Toxicokinetics |
INH6 (0.1-5 μM) showed low cytotoxicity to normal human foreskin fibroblasts (NHF), with cell viability > 90% after 72 hours of treatment at concentrations up to 2 μM [1]
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| References | |||
| Additional Infomation |
INH6 is a novel small molecule inhibitor that targets the Nek2-Hec1 axis, a key regulatory pathway in the process of cell mitosis [1]. Its mechanism of action involves a dual effect: on the one hand, it binds to Nek2 and Hec1, disrupting their interaction; on the other hand, it directly inhibits Nek2 kinase activity, thereby inducing mitotic catastrophe, G2/M phase arrest, and apoptosis in cancer cells [1]. INH6 exhibits broad-spectrum antiproliferative activity against a variety of solid tumor cell lines, and its selectivity for cancer cells is superior to that for normal cells [1]. It is expected to become a lead compound for the development of anticancer drugs targeting mitosis, for the treatment of cervical cancer, breast cancer, lung cancer, and colorectal cancer [1].
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| Molecular Formula |
C19H18N2OS
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| Molecular Weight |
322.42
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| Exact Mass |
322.113
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| CAS # |
1001753-24-7
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| Related CAS # |
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| PubChem CID |
7918451
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.647
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| LogP |
5.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
23
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| Complexity |
398
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
WCZLNJTXHZPHLM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H18N2OS/c1-12-9-13(2)17(14(3)10-12)16-11-23-19(20-16)21-18(22)15-7-5-4-6-8-15/h4-11H,1-3H3,(H,20,21,22)
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| Chemical Name |
N-[4-(2,4,6-Trimethylphenyl)-1,3-thiazol-2-yl]benzamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.75 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1015 mL | 15.5077 mL | 31.0154 mL | |
| 5 mM | 0.6203 mL | 3.1015 mL | 6.2031 mL | |
| 10 mM | 0.3102 mL | 1.5508 mL | 3.1015 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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