Hec1/Nek2
Hec1 (Highly expressed in cancer 1) and Nek2 kinase. The compound disrupts Hec1/Nek2 protein-protein interaction. [1]

T-1101 tosylate exhibits significant in vitro antiproliferative activity (IC50: 14.8–21.5 nM)[1].
T-1101 tosylate disrupts the Hec1/Nek2 protein–protein interaction in the cells[1].
T-1101 tosylate (1μM; 3-24 24 hours) decreases the level of Nek2 in a time-dependent manner[1].
T-1101 tosylate (1 M; 24 hours) causes apoptosis[1].
T-1101 tosylate reduces amounts of cell-cycle related proteins cyclin A1, cyclin B1, and cyclin D1[1].

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T-1101 demonstrated potent antiproliferative activity against various cancer cell lines. The IC50 values were: 21.1 ± 6.1 nM for HeLa, 18.0 ± 0.4 nM for K562, 21.5 ± 0.2 nM for MDA-MB-468, and 14.8 ± 1.2 nM for MDA-MB-231. [1]
T-1101 exhibited potent activity against a panel of breast and liver cancer cells with low nanomolar to submicromolar GI50 values. It also inhibited the growth of U937 (leukemia), COLO 205 (colon cancer), and U2OS (osteosarcoma) cancer cells with GI50 values of 12.4, 29.1, and 83.2 nM, respectively. [1]
T-1101 is active against multi-drug resistant cancer cells including MES-SA/Dx5 (uterine sarcoma) and NCI/ADR-RES (ovarian carcinoma) that are resistant to paclitaxel, and down-regulates the expression of P-glycoprotein in HeLa and MDA-MB-231 cells. [1]
T-1101 shows a synergistic effect with paclitaxel, doxorubicin, and topotecan in several liver cancer cells. [1]
Co-immunoprecipitation assay on K562 cells treated with 100 nM of T-1101 showed a reduced amount of coimmunoprecipitated Hec1 compared to DMSO-treated cells, demonstrating disruption of Hec1/Nek2 protein-protein interaction. The level of Nek2 was lowered in a time-dependent manner in K562 cells treated with T-1101 (1.0 μM). [1]
In MDA-MB-468 cells, T-1101 (1.0 μM) caused mitotic abnormalities including chromosomal misalignment and formation of multipolar spindles. The percentages of mitotic abnormalities increased from 45% at 24 h to 64% at 48 h. [1]
In HeLa cells treated with 1.0 μM of T-1101 for 24 h, the sub-G1 population increased to 20.44% compared to DMSO-treated cells (3.64%). Furthermore, the amount of apoptotic marker proteins cleaved caspase-3 and PARP increased and the amount of antiapoptotic proteins Mcl-1 and XIAP decreased. Amounts of cell-cycle related proteins cyclin A1, cyclin B1, and cyclin D1 were also reduced. [1]
T-1101 was inactive toward non-cancerous cells, a panel of kinases, and hERG, demonstrating cancer specificity, target specificity, and cardiac safety, respectively. [1]

T-1101 tosylate exhibits good oral bioavailability and thermal stability [1].
Oral co-administration of T-1101 tosylate (2.5 mg/kg; p.o.; twice daily) reduces the dose of sorafenib (25 mg/kg to 12.5 mg/kg) needed to exhibit comparable in vivo activity against Huh-7 xenografts [1].
In mice with various human cancer xenografts, T-1101 tosylate (2.5 mg/kg; p.o.; twice daily) exhibits significant in vivo activity[1].

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Oral administration of T-1101 (25 or 50 mg/kg, twice per day) showed significant in vivo activity in mice bearing various human cancer xenografts, including liver cancer cells Huh-7 and breast cancer cells BT-474, MCF-7, and MDA-MB-231. The T/C values were 18-52% at day 28. Specifically, T/C was 18% for Huh-7 (50 mg/kg), 19% for BT-474 (50 mg/kg), 52% for MCF-7 (50 mg/kg), and 40% for MDA-MB-231 (25 mg/kg). [1]
In mice bearing hepatic Huh-7 xenografts, oral co-administration of T-1101 (2.5 mg/kg, twice per day) with sorafenib (12.5 mg/kg, once per day) for 28 consecutive days showed a synergistic effect in vivo, halving the dose of sorafenib required to exhibit comparable in vivo activity. The T/C values were 25% for sorafenib alone (25 mg/kg), 26% for the combination, and 61% for T-1101 alone (2.5 mg/kg). [1]

For coimmunoprecipitation, K562 cells were treated with T-1101 (100 nM), harvested, washed with cold PBS, and lysed on ice in a lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 5 mM EDTA pH 8.0, 0.1% NP-40, 1 mM PMSF, 10 mM NaF, and protease inhibitor cocktail) for 30 min. Lysates were centrifuged at 12,000 rpm at 4°C for 20 min, and the supernatant was incubated with anti-Nek2 antibody or IgG at 4°C for 2.0 h, followed by addition of protein G agarose beads. After mixing at 4°C for 1.0 h, the beads were centrifuged at 12,000 rpm for 20 s, washed 5 times, and boiled in a sample buffer followed by SDS-PAGE. The presence of Hec1 and Nek2 was detected by Western blotting with mouse monoclonal antibodies against Hec1 and Nek2. [1]
Immunoblot analysis of Hec1 and Nek2 protein levels was performed on K562 treated with 1.0 μM of T-1101 at different time points. [1]
For quantification of mitotic abnormalities, MDA-MB-231 cells were treated with 1.0 μM of T-1101 for 24 or 48 h and the percentages of mitotic abnormalities (chromosomal misalignment and multipolar spindles) were calculated by counting the number of abnormal cells from a minimum of 500 randomly selected cells. [1]
For cell cycle analysis, HeLa cells treated with T-1101 (1.0 μM for 24 h) were analyzed by flow cytometry (FACS). [1]

For oral pharmacokinetic studies in mice, T-1101 tosylate and other salts were formulated with 1% CMC in saline for PO dosing at 10 mg/kg. For IV dosing, the free base and salts were formulated with 2% Tween 80 in saline containing 10% DMSO, with a single dose of 1.0 mg/mL administered to healthy male CD-1 mice. [1]
For the tumor tissue distribution study, T-1101 was formulated in 20% hydroxypropyl-β-cyclodextrin (HP-β-CD) in water. SCID mice bearing Huh-7 or MDA-MB-231 xenografts were given a single IV dose of T-1101 at 10 mg/kg. [1]
For the in vivo xenograft combination study with sorafenib, Huh-7 tumor cells were subcutaneously injected into SCID mice. When tumor sizes reached ~100 mm³, mice were treated PO with vehicle control (20% HP-β-CD in H₂O, twice per day), sorafenib alone (25 mg/kg, once per day), T-1101 (2.5 mg/kg, twice per day) with sorafenib (12.5 mg/kg, once per day), or T-1101 alone (2.5 mg/kg, twice per day) for 28 days. T-1101 was formulated in 20% HP-β-CD in H₂O and sorafenib was formulated in 1% carboxymethyl cellulose (CMC) in H₂O. [1]

In mice, oral administration of T-1101 free base (10 mg/kg, formulated with 10% DMSO, 25% PEG200, and 65% saline) had an oral AUC of 137 μM·h. [1]
In rats, T-1101 free base (20 mg/kg, formulated with 5% DMSO, 10% Cremophor EL, and 85% saline) had an IV AUC of 63.4 μM·h and an oral AUC of 533.8 μM·h, with an oral bioavailability (F%) of 84.2% (ClogP 5.07). [1]
In salt screening, T-1101 tosylate (10 mg/kg in 1% CMC in saline) in CD-1 mice had an oral AUC of 62.5 μM·h, Cmax of 8.13 μM, T1/2 of 2.81 h, and oral bioavailability of 77.4%. In beagle dogs, T-1101 tosylate (10 mg/kg) had an oral AUC of 8.61 μM·h, Cmax of 0.98 μM, T1/2 of 4.53 h, and oral bioavailability of 24.9%. [1]
In SCID mice bearing Huh-7 xenografts, IV administration of T-1101 (10 mg/kg) achieved high concentrations in tumor tissues: 3.38, 2.66, 2.07, 0.937, 0.507, and 0.0599 μM at 0.50, 1.0, 4.0, 8.0, 12, and 24 h, respectively. The tumor-to-plasma concentration ratios were 20.7% (30 min), 26.1% (1.0 h), 33.2% (4.0 h), 35.0% (8.0 h), 60.5% (12 h), and 41.1% (24 h). [1]
In MDA-MB-231 tissues, the concentration of T-1101 in the tumors was 2.95 μM and the tumor-to-plasma ratio was 24.1% at 2.0 h. [1]

Central nervous system effects of T-1101 were evaluated on rats using the Irwin test. Behaviors including locomotor activity, restlessness, lethargy, tremors, convulsions, touch response, etc. did not change when the rats were given T-1101 as an oral single dose of up to 60 mg/kg. [1]
For potential cardiopulmonary effects, a study on beagle dogs with an oral single dose of 40 mg/kg did not show any remarkable cardiopulmonary findings. [1]
In a 28-day GLP compliance, repeated-dose toxicology study on rats, no unexpected findings at high oral dose (60 mg/kg) were found in clinical pathology, serum biochemistry, and organ change examinations other than those expected from chemotherapeutic agents. The related symptoms caused by T-1101 in rats were reversible when the administration was discontinued. [1]
T-1101 was inactive towards hERG, indicating cardiac safety. [1]

[1]. Discovery of T-1101 tosylate as a first-in-class clinical candidate for Hec1/Nek2 inhibition in cancer therapy. Eur J Med Chem. 2020 Apr 1;191:112118.

T-1101 tosylate was approved by the US FDA as an investigational new drug at the end of 2016. As of this writing (2020), it was in phase I clinical trials (ClinicalTrials.gov identifiers NCT03195764 and NCT03349073) as an oral drug to evaluate its safety and pharmacokinetics on patients with advanced refractory solid tumors. [1]
T-1101 tosylate is the first-in-class Hec1/Nek2 inhibitor clinical candidate as no such inhibitor had been used or investigated in clinical trials as of this writing. [1]
The docking simulation showed that the calculated binding energies -CDE and -CIE for T-1101 (11.8 and 35.1 kcal/mol) were higher than those of the reference compound 4 (11.7 and 25.7 kcal/mol), indicating stronger binding of T-1101 to Hec1. [1]

C31H31N5O6S3

665.802743196487

665.143

C, 55.92; H, 4.69; N, 10.52; O, 14.42; S, 14.45

2250404-95-4

1438638-83-5;2250404-95-4;

71586372

Light yellow to yellow solid powder

2

12

10

45

836

0

CC1=CC=C(C=C1)S(=O)(=O)O.CC1=CC(=CC(=C1C2=CSC(=N2)NC(=O)C3=CC=NC=C3)C)SC4=NC=C(N=C4)OCCOC

OUJWEAVKLYQREM-UHFFFAOYSA-N

InChI=1S/C24H23N5O3S2.C7H8O3S/c1-15-10-18(34-21-13-26-20(12-27-21)32-9-8-31-3)11-16(2)22(15)19-14-33-24(28-19)29-23(30)17-4-6-25-7-5-17;1-6-2-4-7(5-3-6)11(8,9)10/h4-7,10-14H,8-9H2,1-3H3,(H,28,29,30);2-5H,1H3,(H,8,9,10)

N-[4-[4-[5-(2-methoxyethoxy)pyrazin-2-yl]sulfanyl-2,6-dimethylphenyl]-1,3-thiazol-2-yl]pyridine-4-carboxamide;4-methylbenzenesulfonic acid

T-1101 tosylate; TAI-95 tosylate; T1101; TAI95;T1101 tosylate; TAI95 tosylate; T 1101; TAI 95

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 62.5~100 mg/mL (93.87~150.2 mM)
Ethanol: ~3 mg/mL (~4.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.12 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 20.8 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: ≥ 2.08 mg/mL (3.12 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 20.8 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.

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