Cyclin-dependent kinase 1 (CDK1)
Cyclin-dependent kinase 2 (CDK2)
Cyclin-dependent kinase 4 (CDK4)
Cyclin-dependent kinase 5 (CDK5)
Cyclin-dependent kinase 6 (CDK6)
Cyclin-dependent kinase 9 (CDK9)
Glycogen synthase kinase-3 beta (GSK-3β) (Note: The paper states that in vitro kinase assays showed AT7519 inhibits GSK-3, but the cellular effect observed was activation via dephosphorylation at Ser9) [1]

In MM cells, AT7519 (0-4 μM) TFA causes dose-dependent cytotoxicity with IC50s ranging from 0.5 to 2 μM. This cytotoxicity is linked to GSK-3β activation, which is unrelated to transcriptional inhibition. AT7519 TFA defeats both the BMSC's protective function and the proliferative advantage brought about by cytokines. MM cells undergo time-dependent apoptosis in response to AT7519 (0.5 μM) TFA. Furthermore, AT7519 (0.5 μM) TFA partially inhibits RNA synthesis in MM.1S cells and inhibits the phosphorylation of RNA polymerase II CTD[1]. In human tumor cell lines, AT7519 (250 nM) TFA inhibits the progression of the cell cycle. Human tumor cell lines are also induced to undergo apoptosis by AT7519 TFA[2]. TFA AT7519 (100-700 nM) causes leukemia cell lines to undergo apoptosis. Furthermore, transcription in human tumor cell lines is inhibited by AT7519TFA. Moreover, AT7519 TFA lowers levels of antiapoptotic proteins and inhibits RNA polymerase II[3].

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AT7519 demonstrated dose-dependent cytotoxicity against multiple myeloma (MM) cell lines (both therapy-sensitive and resistant) and primary patient-derived MM cells, with IC50 values ranging from 0.5 to 2 μM at 48 hours. It showed minimal cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. [1]
AT7519 partially inhibited the proliferative advantage conferred to MM cells by bone marrow stromal cells (BMSCs) and cytokines (IL-6, IGF-1) in co-culture assays. [1]
AT7519 induced cell cycle arrest (increase in G0/G1 and G2/M phases) and apoptosis in MM.1S cells in a time- and dose-dependent manner, as shown by cell cycle analysis (sub-G1 population), Annexin V/PI staining, and cleavage of caspases -9, -3, and -8. [1]
AT7519 treatment led to the rapid dephosphorylation (within 1 hour) of the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) at both Serine 2 and Serine 5, without affecting total RNA pol II protein levels. This correlated with approximately 50% inhibition of total RNA synthesis, as measured by [³H] uridine incorporation after 48 hours. [1]
AT7519 treatment resulted in decreased expression of the short-lived anti-apoptotic proteins Mcl-1 and XIAP within 4 hours. [1]
AT7519 induced rapid dephosphorylation (activation) of GSK-3β at Serine 9 within 2 hours, accompanied by increased phosphorylation of its downstream substrate glycogen synthase. This activation was associated with downregulation of GSK-3β targets c-Myc and cyclin D1. The activation of GSK-3β occurred independently of its upstream regulators (Akt, p70S6K), which were paradoxically phosphorylated (activated). [1]
Pretreatment with the selective GSK-3 inhibitor AR-A014418 or knockdown of GSK-3β using specific shRNAs partially rescued MM cells from AT7519-induced cytotoxicity, indicating that GSK-3β activation contributes to the apoptotic effect. [1]
Treatment with alpha-amanitin, a specific RNA pol II inhibitor, recapitulated the RNA pol II dephosphorylation but did not affect GSK-3β phosphorylation status, demonstrating that GSK-3β activation by AT7519 is independent of transcriptional inhibition. [1]

In a mouse model of human MM xenograft, AT7519 TFA inhibits the growth of tumors[1]. Early-stage HCT116 tumor xenografts are inhibited in their growth by AT7519 (4.6 and 9.1 mg/kg/dose). Target CDKs are also inhibited by AT7519 (10 mg/kg, i.p.) TFA in BALB/c nude mice harboring HCT116 tumors[2].

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In a human MM.1S xenograft mouse model, intraperitoneal administration of AT7519 (15 mg/kg) significantly inhibited tumor growth compared to the vehicle control group. [1]
Treatment with AT7519 (15 mg/kg, either once daily for five days over two weeks or three times per week for four weeks) significantly prolonged the median overall survival of tumor-bearing mice (40 and 39 days, respectively) compared to the control group (27.5 days). [1]
Immunohistochemical analysis of tumors from AT7519-treated mice showed increased activation of caspase-3, confirming induction of apoptosis in vivo. [1]
AT7519 treatment did not significantly affect the body weight of the treated mice, suggesting tolerability at the administered doses in this model. [1]

Kinase assays using radiometric filter binding are conducted for CDK1, CDK2, and GSK3-β. The format of the assays is ELISA for CDKs 4 and 6, and DELFIA for CDK 5. The relevant CDK and 0.12 μg/mL Histone H1 are incubated for 2 or 4 hours, respectively, in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/mL BSA, 45 μM ATP (0.78 Ci/mmol), and various concentrations of AT7519. In order to test GSK3-β, the appropriate enzyme and 5 μM glycogen synthase peptide 2 are added, and the mixture is incubated for three hours at 10 mM MOPS pH 7.0, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, 0.2 mM EDTA, 10 mM MgCl2, 0.01% β-mercaptoethanol, 15 μM ATP (2.31 Ci/mmol), all of which are tested. Millipore MAPH filter plates are used to filter the assay reactions after an excess of orthophosphoric acid is added to stop the reaction. After that, the plates are cleaned, scintillant is added, and radioactivity is determined using a Packard TopCount scintillation counting device. For a duration of 30 minutes, CDK5, CDK5/p35, 1μM of a biotinylated Histone H1 peptide (Biotin-PKTPKKAKKL), pH 7.5, 25 mM Tris-HCl, 0.025% Brij-35, 0.1 mg/mL BSA, 1 mM DTT, 15 μM ATP, and various concentrations of AT7519 are incubated. Time-resolved fluorescence at λex=335nm, λem=620nm is used to stop the assay reactions using EDTA, transfer the mixture to Neutravidin-coated plates, and quantify the phosphorylated peptide using a rabbit phospho-cdk1 substrate polyclonal antibody and DELFIA europium-labelled anti-rabbit IgG secondary antibody. Plates are coated with GST-pRb769-921 and blocked with Superblock for the CDK 4 and 6 assays. In order to initiate the reaction, ATP is added to CDK4 or 6. The incubation conditions include 15 mM MgCl2, 50 mM HEPES, pH 7.4, 1 mM DTT, 1 mM EGTA, pH 8.0, 0.02% Triton X-100, 2.5% DMSO, and various concentrations of AT7519. Reactions are halted by adding 0.5 M EDTA pH 8.0 after 30 minutes. After that, plates are cleaned and incubated for one hour with a secondary antibody (alkaline phosphatase linked anti-rabbit) and another hour with the primary antibody (anti-p-Rb Serine 780) diluted in Superblock. Fluorescence is measured on a Spectramax Gemini plate reader at excitation of 450 nm and emission of 580 nm after plates are developed using the Attophos system. Using GraphPad Prism software, IC50 values are computed from replicate curves in every scenario.

The assessable effects of AT7519 on the viability of primary MM cells, MM cell lines, and PBMNCs are determined by measuring the dye absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrasodium bromide (MTT). The assay for measuring DNA synthesis uses tritiated thymidine uptake (3H-TdR). 3H-TdR incorporation is measured after MM cells (2–3 × 10⁴ cells/well) are cultured for 24 or 48 hours at 37°C in 96-well culture plates with media and varying concentrations of AT7519 and/or recombinant IL-6 (10 ng/mL) or IGF-1 (50 ng/mL).

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Cell viability assay (MTT assay): MM cell lines, primary patient MM cells, or peripheral blood mononuclear cells were cultured in 96-well plates with increasing concentrations of AT7519 (0-4 μM) for 24, 48, or 72 hours. The tetrazolium dye MTT was added, and the absorbance was measured to determine cell viability. [1]
DNA synthesis/proliferation assay: MM cells were cultured with AT7519 and/or cytokines (IL-6, IGF-1) for 24 or 48 hours. Tritiated thymidine (³H-TdR) was added for the final 8-16 hours of culture. Cells were harvested, and incorporated radioactivity was measured using a scintillation counter. [1]
RNA synthesis assay: MM.1S cells were incubated with AT7519 (0.5 μM) for various time points (4-48 hours). During the last 3.5 hours, [³H] uridine was added to the culture. Cells were harvested, and incorporated radioactivity was measured to assess RNA synthesis. [1]
Cell cycle analysis: MM cells treated with AT7519 were harvested, fixed with ethanol, treated with RNase, and stained with propidium iodide (PI). DNA content was analyzed by flow cytometry to determine cell cycle distribution. [1]
Apoptosis detection (Annexin V/PI staining): MM cells treated with AT7519 were harvested, stained with Annexin V and PI, and analyzed by flow cytometry. The percentage of apoptotic cells was defined as the sum of Annexin V-positive/PI-negative (early apoptosis) and Annexin V-positive/PI-positive (late apoptosis) cells. [1]
Western blotting: MM cells treated with AT7519 were lysed. Protein lysates were separated by SDS-PAGE, transferred to membranes, and probed with specific primary antibodies against targets of interest (e.g., phospho-RNA pol II, total RNA pol II, phospho-GSK-3β, total GSK-3β, caspases, Mcl-1, XIAP, cyclins, CDKs) and appropriate secondary antibodies. Protein bands were visualized using enhanced chemiluminescence. [1]
Lentiviral shRNA knockdown: Lentiviral particles containing GSK-3β-specific shRNA or control shRNA were produced in 293T cells. MM.1S cells were infected with the viral supernatant in the presence of polybrene. After selection with puromycin, successful knockdown was confirmed by western blotting. These cells were then used in viability assays to assess the role of GSK-3β in AT7519-induced cytotoxicity. [1]

In order to assess the in vivo anti-MM activity of AT7519, 5×10⁶ MM.1S cells are subcutaneously injected into male SCID mice using 100 μL of serum-free RPMI 1640 medium. Mice are treated intraperitoneally (IP) with vehicle or AT7519 dissolved in 0.9% saline solution when tumors are detectable. Ten mice in the first group receive a daily dose of 15 mg/kg for two weeks, while the second group receives a daily dose of 15 mg/kg three times a week for four weeks in a row. At the same time, the carrier is given to the control group alone. Tumor volume is calculated using the formula V= 0.5 a × b², where a represents the tumor's long diameter and b its short diameter. Tumor size is measured every other day in two dimensions using calipers. When a tumor is ulcerated or grows to a size of 2 cm³, the animal is killed. From the first day of treatment until death, survival and tumor growth are assessed.

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MM xenograft model: Severe combined immunodeficient (SCID) mice were inoculated subcutaneously with 5×10⁶ MM.1S cells. When tumors became measurable, mice were randomized into treatment groups. [1]
AT7519 was dissolved in saline (0.9%) for administration. [1]
Two dosing regimens were tested via intraperitoneal (IP) injection: 1) 15 mg/kg once daily for five consecutive days, repeated after a two-day break for a second week (total of 10 doses over 2 weeks). 2) 15 mg/kg once daily, three times per week (e.g., Monday, Wednesday, Friday) for four consecutive weeks. [1]
The control group received vehicle (saline) alone on the same schedule. [1]
Tumor dimensions were measured regularly with calipers, and volume was calculated. Animals were monitored for survival and body weight. [1]

[1]. AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition. Oncogene. 2010 Apr 22;29(16):2325-36.

[2]. Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines. Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell

[3]. AT7519, a cyclin-dependent kinase inhibitor, exerts its effects by transcriptional inhibition in leukemia cell lines and patient samples. Mol Cancer Ther. 2010 Apr;9(4):920-8.

AT7519 is a novel small-molecule multi-cyclin-dependent kinase (CDK) inhibitor with the chemical name N-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxamide. [1] This study focuses on its preclinical evaluation in multiple myeloma (MM). Dysregulation of CDKs and cyclins is a hallmark of MM, making it an attractive therapeutic target. [1] In MM, AT7519 has a dual mechanism of action: 1) inhibiting transcriptional CDKs (e.g., CDK9, CDK7), leading to dephosphorylation of RNA polymerase II CTD, transcriptional repression, and downregulation of short-lived pro-survival proteins (e.g., Mcl-1 and XIAP). 2) promoting apoptosis in a transcription-independent manner by directly or indirectly activating GSK-3β through dephosphorylation at the Ser9 site. Given the inhibitory effect shown by in vitro kinase activity assays, this effect on GSK-3β is surprising. [1]
This study provides a theoretical basis for the clinical evaluation of AT7519 in patients with multiple myeloma (MM). [1]

C18H18CL2F3N5O4

496.2678

495.068

C, 43.56; H, 3.66; Cl, 14.29; F, 11.48; N, 14.11; O, 12.90

1431697-85-6

AT7519;844442-38-2;AT7519 Hydrochloride;902135-91-5

71576690

Solid powder

5

9

4

32

563

0

ClC1C([H])=C([H])C([H])=C(C=1C(N([H])C1C([H])=NN([H])C=1C(N([H])C1([H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H])=O)=O)Cl.FC(C(=O)O[H])(F)F

XTOQTRBZWZONHQ-UHFFFAOYSA-N

InChI=1S/C16H17Cl2N5O2.C2HF3O2/c17-10-2-1-3-11(18)13(10)15(24)22-12-8-20-23-14(12)16(25)21-9-4-6-19-7-5-9;3-2(4,5)1(6)7/h1-3,8-9,19H,4-7H2,(H,20,23)(H,21,25)(H,22,24);(H,6,7)

4-[(2,6-dichlorobenzoyl)amino]-N-piperidin-4-yl-1H-pyrazole-5-carboxamide;2,2,2-trifluoroacetic acid

AT7519 trifluoroacetate; AT-7519 trifluoroacetate; AT 7519 trifluoroacetate

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 : ~100 mg/mL (~201.50 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.04 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: ≥ 2.5 mg/mL (5.04 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.04 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.

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