INH154 is particularly successful in treating breast cancers with increased expression of both Hec1 and Nek2. INH154 is the most effective tumor cell growth inhibitor. The IC50 values of INH154 in HeLa and MDA-MB-468 cancer cells were 0.20 and 0.12 μM, respectively. INH154 also suppresses the growth of leukemia, osteosarcoma, and glioblastoma cells [1].

INH154 is particularly successful in treating breast cancers with increased expression of both Hec1 and Nek2. INH154 is the most effective tumor cell growth inhibitor. The IC50 values of INH154 in HeLa and MDA-MB-468 cancer cells were 0.20 and 0.12 μM, respectively. INH154 also suppresses the growth of leukemia, osteosarcoma, and glioblastoma cells [1].

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INH154 exhibited potent growth inhibitory effects on various cancer cell lines with IC₅₀ values of 0.20 μM in HeLa cells, 0.12 μM in MDA-MB-468 cells, and also showed efficacy against leukemia (K562), osteosarcoma (U2OS), and glioblastoma (T98G) cells. In contrast, it had no significant growth inhibitory effect on non-tumorigenic fibroblast (HS27) and mammary epithelial (MCF10A) cells. [1]
Colony formation assays confirmed that INH154 effectively inhibited the growth of HeLa and MDA-MB-468 cancer cells in a dose-dependent manner. [1]
Treatment with INH154 induced mitotic catastrophe in cancer cells, characterized by increased chromosomal misalignment and multipolar spindle configurations in a time-dependent manner. [1]
Flow cytometry analysis using Annexin-V/Propidium Iodide staining showed that INH154 treatment (1 μM, 48 hours) significantly increased the percentage of apoptotic and necrotic cells (14.7% apoptotic, specific percentage for necrosis not separately quantified) compared to DMSO-treated controls. [1]
Biotin-conjugated INH154 specifically pulled down wild-type Hec1 protein but failed to pull down Hec1 mutants (W395A or WLK/AAA) or other proteins like Nek2 and Nuf2 in affinity pull-down assays, confirming its specific binding to Hec1. [1]
Cells stably expressing Hec1 mutants (W395A or WLK/AAA), which are deficient in INH154 binding, showed significantly elevated IC₅₀ values for INH154, indicating acquired resistance. [1]
Treatment with INH154 (1 μM) triggered a time-dependent, proteasome-mediated degradation of Nek2 protein (over 95% reduction after 18 hours) without affecting Nek2 mRNA levels. Co-treatment with the proteasome inhibitor MG132 prevented Nek2 degradation. [1]
INH154 treatment abolished the phosphorylation of Hec1 at serine 165 (pS165), a modification dependent on Nek2 kinase activity, as shown by Western blot and immunofluorescence staining at kinetochores. [1]
The induction of Nek2 degradation by INH154 required the physical interaction between Hec1 and Nek2. Cells expressing Hec1 mutants defective in Nek2 binding (Δ3, lacking amino acids 408-422) or Nek2 mutants defective in Hec1 binding (R361L) were resistant to INH154-induced Nek2 degradation. [1]
The proposed mechanism is that INH154 binding to Hec1 creates a "death-trap" conformation that, when bound by Nek2, triggers a conformational change in Nek2 leading to its proteasomal degradation. [1]

In a dose-dependent way, tumor growth was markedly slowed in mice treated with INH154 compared to control animals. Tumor proliferation index evaluated by BrdU staining showed a significant reduction in residual tumor under treatment with INH154 when compared to vehicle alone, which is consistent with the tumor growth results. In comparison to tumors treated with a vehicle, INH154-treated tumors exhibited considerably decreased expression levels of Nek2 and Hec1 S165 phosphorylation. However, there was hardly any change between the treatment and control groups when the mice's body weight was assessed during the 6.5-week treatment period. Additionally, when high-dose INH154 (20 mg/kg) was used to treat INH toxicity in normal BALB/c ByJNarl mice, there were no discernible changes in these animal groups' body weight, blood chemistry, or complete blood count (CBC) analysis [1].

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In a xenograft mouse model using MDA-MB-468 triple-negative breast cancer cells (which express high levels of Hec1 and Nek2), intraperitoneal administration of INH154 at 5 mg/kg or 20 mg/kg, thrice weekly for 6.5 weeks, significantly suppressed tumor growth in a dose-dependent manner compared to vehicle-treated controls. [1]
Immunohistochemical analysis of residual tumors from INH154-treated mice showed a clear reduction in the proliferation index (BrdU staining), substantial reduction in Nek2 protein levels, and diminished phosphorylation of Hec1 at S165 compared to vehicle-treated tumors. [1]

For cell viability/anti-proliferative assays (XTT assay), cells were plated in 96-well dishes one day prior to treatment. Cells were then exposed to a range of concentrations of INH154 for 4 days. Cell viability was measured, and IC₅₀ values were determined from the dose-response curves. [1]
For colony formation assays, HeLa or MDA-MB-468 cells were treated with various doses of INH154. After a period of incubation allowing colony formation, colonies were stained and counted to assess the inhibitory effect on long-term cell proliferation. [1]
To analyze mitotic defects, cells treated with INH154 were fixed and stained. Chromosome alignment was assessed using DNA stain (Hoechst 33342). Spindle polarity was evaluated by immunofluorescence staining for γ-tubulin (centrosomes), α-tubulin (microtubules), and DAPI (chromosomes). The percentage of cells with misaligned chromosomes or multipolar spindles was quantified. [1]
Apoptosis was assessed by flow cytometry. Cells treated with INH154 were stained with Annexin V and Propidium Iodide (PI) according to standard protocols. The percentages of cells in early apoptosis (Annexin V-positive, PI-negative) and late apoptosis/necrosis (Annexin V-positive, PI-positive) were determined. [1]
To study protein degradation and phosphorylation, cells were treated with INH154 for specified durations. For proteasome inhibition experiments, cells were co-treated with INH154 and MG132. Cells were then lysed, and protein levels were analyzed by Western blot using specific antibodies against Nek2, phospho-S165 Hec1, total Hec1, and loading controls. [1]
For binding specificity studies (biotin pull-down), cell extracts expressing GFP-tagged Hec1 or its mutants were prepared in lysis buffer. The clarified lysate was incubated with neutravidin resin conjugated to biotin-INH154. After extensive washing, bound proteins were eluted and analyzed by Western blot with anti-GFP antibody. [1]
For co-immunoprecipitation to study protein-protein interactions, cells co-expressing tagged proteins (e.g., GFP-Hec1 and Myc-Nek2) were lysed. Lysates were incubated with antibodies against the tag (e.g., anti-GFP) followed by Protein G Sepharose. Immunoprecipitates were washed, eluted, and analyzed by Western blot with relevant antibodies. [1]

An athymic nude mouse xenograft model was established by injecting MDA-MB-468 breast cancer cells (2 × 10⁶ cells) into the mammary fat pads of 6- to 8-week-old female nude mice. When tumors reached approximately 100 mm³ in volume, mice were randomly divided into treatment groups (n=6-7 per group). [1]
INH154 was formulated in a vehicle consisting of 5% DMSO, 7.5% ethanol, 7.5% Cremophor EL, 20% PEG400, and 60% saline. [1]
Mice received intraperitoneal (i.p.) injections of the vehicle, 5 mg/kg INH154, or 20 mg/kg INH154. Injections were administered three times per week. [1]
Treatment continued for 6.5 weeks. Mouse body weights and tumor dimensions (length and width) were measured twice weekly. Tumor volume was calculated using the formula (length × width²)/2. [1]
One week after the final injection, mice were sacrificed. Tumors were harvested, fixed, and processed for immunohistochemical analysis of BrdU incorporation (proliferation), Nek2 expression, and phospho-S165 Hec1 levels. [1]

In a 6.5-week xenotransplantation efficacy study, mice treated with 5 mg/kg or 20 mg/kg INH154 showed minimal weight difference compared to the vector control group, indicating no significant systemic toxicity was observed at these effective doses. [1] In another toxicity assessment, normal BALB/c ByJNarl mice were treated with a high dose of INH154 (20 mg/kg). No significant differences were observed in weight, blood biochemistry, or complete blood cell count (CBC) analysis compared to the control group, indicating very low or no toxicity. [1]

[1]. Novel small molecules disrupting Hec1/Nek2 interaction ablate tumor progression by triggering Nek2 degradation through a death-trap mechanism. Oncogene. 2015 Mar 5;34(10):1220-30.

INH154 is a third-generation INH (Nek2 and Hec1 binding inhibitor) small molecule derivative whose structure has been optimized based on INH1 and INH41 to improve efficacy. [1]
Analysis of gene expression databases of breast cancer patients showed that co-expression of Hec1 and Nek2 was highly correlated and associated with the worst distant metastasis progression-free survival (DMFS) and recurrence-free survival (RFS). This provides a theoretical basis for targeting the Hec1/Nek2 interaction in cancers with this characteristic. [1]
Its mechanism of action involves a novel “death trap” model: INH154 binds to Hec1, and the drug-bound Hec1 interface then induces Nek2 into the complex. The subsequent binding induces a conformational change in Nek2, causing it to be degraded by the proteasome, while blocking its kinase activity on Hec1 S165. [1]
Compared to the first-generation compound INH1, INH154 is approximately 100 times more effective at inhibiting cancer cell growth. [1]

C22H24N4OS

392.517163276672

392.167

1587705-63-2

91669222

Light yellow to yellow solid powder

4.4

1

5

4

28

509

0

S1C(NC(C2C=CN=CC=2)=O)=NC(=C1)C1C(C)=CC(=CC=1C)N1CCCCC1

BBVORAZSFSZSKG-UHFFFAOYSA-N

InChI=1S/C22H24N4OS/c1-15-12-18(26-10-4-3-5-11-26)13-16(2)20(15)19-14-28-22(24-19)25-21(27)17-6-8-23-9-7-17/h6-9,12-14H,3-5,10-11H2,1-2H3,(H,24,25,27)

N-[4-(2,6-dimethyl-4-piperidin-1-ylphenyl)-1,3-thiazol-2-yl]pyridine-4-carboxamide

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 : ~83.33 mg/mL (~212.29 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (15.92 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 62.5 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.

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Solubility in Formulation 2: 2.5 mg/mL (6.37 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 2.5 mg/mL (6.37 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (5.30 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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