Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) - Inhibitor. Haspin is a protein kinase that specifically phosphorylates histone H3 at threonine 3 (H3T3ph), which is necessary for mitosis progression [1]
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- IC50 value for Haspin enzyme inhibition: 2 nM (determined by FRET assay in a panel of 27 protein kinases) [1]
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- IC50 values for cell proliferation inhibition:
- HCT-116 (colon cancer): 500 nM [1]
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- HeLa (cervical cancer): 473 nM [1]
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- MDA-MB-231 (breast cancer): 752 nM [1]
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- Wi-38 (normal human diploid fibroblasts): 1059 nM [1]
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The growth of dyes such as HCT-116, HeLa, MDA-MB-231, and Wi-38 cells is dose-dependently inhibited by CHR-6494 (0-10-5 nM; 72 hours); the IC50 of 500 nM and 473 nM CHR, respectively, are produced by CHR-6494 (500 nM). Additionally, the spindle assembly checkpoint protein BUB1 and the mitotic division marker cyclin B1, 752 nM and 1059 nM, are upregulated by CHR-6494 (500 nM). With an IC50 ranging from 396 nM to 1229 nM, CHR-6494 exhibits inhibitory effect against melanoma cell lines, including BRAFV600E mutants, NRAS mutants, and wild-type cells [2]. 300 nM and 600 nM of CHR-6494. ; 72 hours) causes a cell exchange that increases the activity of caspase 3/7 by three and six times in COLO-792 cells, and eighty-five and sixteen times in RPMI-7951 cells [2]. When CHR-6494 and MEK dye are combined, they can effectively suppress melanoma cell viability, accelerate melanoma cell shutdown, individually control the course of the cell cycle by stopping melanoma cells at various stages, and stop melanoma cell migration [2]. The anti-proliferative activity of MLN8237 on MDA-MB-231, SKBR3 breast cancer cells is enhanced by CHR-6494 (50, 200 nM; 1 week) [3]. The combination of MLN8237 and CHR-6494 (200 nM; 72 hours) improves the sterility of SKBR3 and MDA-MB-231 cells [3].

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Inhibition of Histone H3T3 Phosphorylation: CHR-6494 inhibited H3T3 phosphorylation in a dose-dependent manner in HeLa, HCT-116, and MDA-MB-231 cancer cell lines, as demonstrated by western blot and immunofluorescence. At 500 nM concentration, the H3T3ph signal was extremely low. The compound did not affect total Haspin protein levels nor phosphorylation of other histone H3 residues (H3S10ph and H3S28ph) [1]
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- Anti-proliferative Effects: CHR-6494 inhibited cancer cell growth in a dose-dependent manner as measured by XTT assay. IC50 values ranged from 473-752 nM in cancer cells, while normal Wi-38 cells showed approximately 2-fold higher IC50 (1059 nM), indicating selectivity for cancer cells. Overexpression of Haspin partially rescued cells from the effects of the compound [1]
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- Cell Cycle Arrest: CHR-6494 treatment caused dose-dependent G2/M phase arrest. The percentage of cells in G2/M increased from basal 8-15% to 30-50% upon drug treatment. In HCT-116 cells, the G2/M arrest persisted for 24-48 hours after drug withdrawal [1]
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- Apoptosis Induction: CHR-6494 induced apoptosis in a dose-dependent manner as shown by Annexin V-FITC/PI staining. Apoptotic effect ranged from 89.88% in HeLa cells, 63.27% in HCT-116 cells, to 30.41% in MDA-MB-231 cells [1]
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- Mitotic Catastrophe: CHR-6494 treatment caused mitotic defects including metaphase misalignment, abnormal multi-polar spindle morphology, and centrosome amplification. The percentage of anaphase cells decreased in a dose-dependent manner. The number of cells with >2 centrosomes increased with drug concentration [1]
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- Spindle Checkpoint Activation: CHR-6494 treatment upregulated the spindle assembly checkpoint protein BUB1 and the mitotic arrest marker cyclin B1 in all three cancer cell lines, indicating activation of mitotic checkpoints [1]
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- Kinase Selectivity: In a panel of 27 protein kinases tested using FRET-based Z'-LYTE Kinase Assay, CHR-6494 showed IC50 of 2 nM for Haspin, while IC50 values for all other kinases were in the micromolar range, demonstrating high selectivity [1]
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In nude mice of the human colorectal colon, HCT-116 was shown to inhibit tumor growth in CHR-6494 (50 mg/kg; intraperitoneal injection; 2 cycles of 5 consecutive days, 15 d) without significantly changing body weight [1]. In nude mice carrying MDA-MB-231 xenograft tumors, CHR-6494 (20 mg/kg; intraperitoneal injection for 15 consecutive days) has an inhibitory effect on tumor volume and weight when compared with cosmetics [3]. (20 mg/kg; po) for 15 days straight intraperitoneal administration) increases the suppression of tumor weight and volume in vivo by MLN8237 (20 mg/kg; po) [3].

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Antitumor Activity in Xenograft Model: The antitumor activity of CHR-6494 was evaluated using HCT-116 human colorectal cancer cells xenografted in nude mice. Mice with established tumors (average volume 200 mm³) were treated with 50 mg/kg CHR-6494 by intraperitoneal injection daily for two cycles of five consecutive days over 15 days. Dose-dependent tumor growth inhibition was demonstrated. Upon cessation of treatment, tumors started to regrow, confirming that the anti-proliferative effect was caused by the compound [1]
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- Safety Profile: Histopathological analysis of colon, liver, and kidney sections from treated mice revealed no abnormalities. Body weight of CHR-6494-treated mice did not change during the treatment period, implying lack of toxicity under the described conditions [1]
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High-Throughput Screening for Haspin Inhibitors: A radiometric enzymatic Haspin kinase assay was developed based on incorporation of radioactivity from [γ-³³P]-ATP into histone H3 in a homogeneous FlashPlate 384-well format. The assay used 10 ng Haspin enzyme and 1.5 μg histone H3 substrate per well. The reaction was initiated by adding [γ-³³P]-ATP, incubated for 75 minutes at room temperature, stopped with EDTA, washed, and counted in a TopCount Counter [1]
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- Kinase Selectivity Profiling: The enzymatic inhibitory capacity of CHR-6494 was tested against a panel of 27 protein kinases using FRET-based Z'-LYTE Kinase Assay. This assay is based on differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. In the primary reaction, the kinase transfers phosphate from ATP to a synthetic FRET peptide. In the secondary reaction, a site-specific protease cleaves non-phosphorylated FRET peptides, disrupting FRET between donor and acceptor fluorophores. Phosphorylated peptides maintain FRET. The ratio of donor to acceptor emission after excitation at 400 nm quantifies reaction progress [1]
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Cell Viability Assay (XTT): Cells were seeded in 94-well plates at 4×10⁴ cells per well and allowed to attach for 24 hours. Medium was then replaced with medium containing different concentrations of CHR-6494 (0.001-10 μM). After 72 hours, XTT reagent was added and optical density was measured. IC50 values were determined using GraphPad Prism software [1]
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- Apoptosis Assay (Annexin V-FITC/PI): Cells were treated with CHR-6494 (0.5, 1, 1.5 μM) or DMSO control for 48 hours. Floating and attached cells were collected, washed with PBS, and stained with Annexin V-FITC and propidium iodide according to manufacturer's instructions. Stained cells were analyzed by flow cytometry [1]
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- Cell Cycle Analysis: Cells were fixed in ice-cold 70% ethanol overnight at -20°C, washed, resuspended in phosphate-citrate buffer, and stained with 25 μg/mL propidium iodide containing 50 μg/mL RNase A for 30 minutes at 37°C. DNA content was analyzed by flow cytometry [1]
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- Western Blot Analysis: Cells treated with 500 nM CHR-6494 or DMSO control for 48 hours were harvested, lysed, and proteins separated by SDS-PAGE. After transfer to nitrocellulose membranes, proteins were detected using antibodies against H3T3ph, total H3, Haspin, H3S28ph, H3S10ph, tubulin, cyclin B1, BUB1, and actin. Membranes were scanned and quantified using Odyssey IR scanner [1]
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- Immunofluorescence Staining: Cells grown on coverslips were treated, fixed with 4% paraformaldehyde or cold methanol, permeabilized, and incubated with primary antibodies against H3T3ph, α-tubulin, γ-tubulin, or H3S10ph. After washing, cells were incubated with Alexa 488 and Alexa 568-labeled secondary antibodies, mounted with DAPI, and imaged using confocal microscopy [1]
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Xenograft Study Design:** Athymic nu/nu male mice (4-5 weeks old) were injected subcutaneously with 3.5×10⁶ HCT-116 cells in 250 μL sterile PBS. When tumors reached average volume of 200 mm³ (15 days after injection), 24 mice with homogeneous tumor sizes were randomized into two groups: control group (n=8) treated with vehicle (10% DMSO/20% 2-hydroxypropyl-β-cyclodextrin) and treatment group (n=16) treated with 50 mg/kg CHR-6494 dissolved in the same vehicle. Treatment was administered daily by intraperitoneal injection in two cycles of five consecutive days over 15 days. Tumor dimensions and body weight were measured twice weekly. Tumor volume was calculated as V = D × d²/2, where D is long axis and d is short axis [1]
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- **Toxicity Assessment:** Upon sacrifice, colon, lung, liver, and kidney tissues were collected, fixed, and stained with hematoxylin and eosin for histological analysis to assess endogenous toxicity [1]
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Xenograft Study Design: Athymic nu/nu male mice (4-5 weeks old) were injected subcutaneously with 3.5×10⁶ HCT-116 cells in 250 μL sterile PBS. When tumors reached average volume of 200 mm³ (15 days after injection), 24 mice with homogeneous tumor sizes were randomized into two groups: control group (n=8) treated with vehicle (10% DMSO/20% 2-hydroxypropyl-β-cyclodextrin) and treatment group (n=16) treated with 50 mg/kg CHR-6494 dissolved in the same vehicle. Treatment was administered daily by intraperitoneal injection in two cycles of five consecutive days over 15 days. Tumor dimensions and body weight were measured twice weekly. Tumor volume was calculated as V = D × d²/2, where D is long axis and d is short axis [1]
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- Toxicity Assessment: Upon sacrifice, colon, lung, liver, and kidney tissues were collected, fixed, and stained with hematoxylin and eosin for histological analysis to assess endogenous toxicity [1]
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In Vivo Toxicity: In xenografted nude mice treated with 50 mg/kg CHR-6494 intraperitoneally for 15 days, no abnormalities were observed in colon, liver, and kidney tissues by histopathological analysis. Body weight of treated mice did not change during the treatment period, indicating lack of overt toxicity under the described conditions [1]
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- Selectivity for Cancer Cells: CHR-6494 showed approximately 2-fold higher IC50 in normal Wi-38 human diploid fibroblasts (1059 nM) compared to cancer cell lines (473-752 nM), suggesting preferential activity against cancer cells [1]
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[1]. Antitumor activity of a small-molecule inhibitor of the histone kinase Haspin. Oncogene. 2012 Mar 15;31(11):1408-18.

[2]. Anti-Melanoma Activities of Haspin Inhibitor CHR-6494 Deployed as a Single Agent or in a Synergistic Combination with MEK Inhibitor. J Cancer. 2017 Aug 25;8(15):2933-2943.

[3]. CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A inhibitor resistance in breast cancer. Cancer Commun (Lond). 2021 Feb;41(2):121-139.

Chemical Identity: CHR-6494 is 3-(1H-indazol-5-yl)-N-propylimidazo[1,2-b]pyridazin-6-amine. It was identified from a library of 117,995 compounds through high-throughput screening for Haspin inhibitors [1]
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- First-in-Class Haspin Inhibitor: CHR-6494 is characterized as a first-in-class inhibitor of the histone kinase Haspin with a wide spectrum of anticancer effects in vitro, ex vivo, and in vivo [1]
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- Mechanism of Action: By inhibiting Haspin, CHR-6494 blocks phosphorylation of histone H3 at threonine 3 (H3T3ph), which is required for proper chromatid cohesion, metaphase alignment, and normal progression through mitosis. This leads to mitotic catastrophe characterized by spindle abnormalities, centrosome amplification, G2/M arrest, and subsequent apoptosis [1]
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- Anti-angiogenic Activity: In ex vivo chicken embryo aortic arch ring assays, CHR-6494 at 1 μM concentration reduced bFGF-induced sprouting vessel area by 70%, demonstrating anti-angiogenic properties [1]
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- Comparison with Other Mitotic Kinase Inhibitors: CHR-6494 belongs to the new generation of anti-mitotic compounds that target components of the mitotic machinery other than microtubules, potentially avoiding adverse effects associated with taxanes. Unlike Aurora B kinase inhibitors, CHR-6494 did not increase polyploidy levels [1]
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- Clinical Potential: Haspin is overexpressed in lymphomas, suggesting that CHR-6494 may have particular utility in hematological malignancies. The compound merits further preclinical development including studies on bioavailability, pharmacokinetics, and additional cancer models [1]
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- Source: CHR-6494 was supplied by Chroma Therapeutics (Oxfordshire, UK) [1]
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C16H16N6

292.346

292.143

1333377-65-3

CHR-6494 TFA;1458630-17-5

70679308

Light yellow to khaki solid powder

1.4±0.1 g/cm3

1.747

2.48

2

4

4

22

378

0

CZZCAOGIEGXMBZ-UHFFFAOYSA-N

InChI=1S/C16H16N6/c1-2-7-17-15-5-6-16-18-10-14(22(16)21-15)11-3-4-13-12(8-11)9-19-20-13/h3-6,8-10H,2,7H2,1H3,(H,17,21)(H,19,20)

3-(1H-indazol-5-yl)-N-propylimidazo[1,2-b]pyridazin-6-amine

CHR-6494 CHR6494 CHR 6494

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 : ~50 mg/mL (~171.03 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.55 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.

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Solubility in Formulation 2: 1 mg/mL (3.42 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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