AT7519 Hydrochloride (AT7519 HCl) is a multi-CDK inhibitor for CDK1, 2, 4, 6 and 9.

CDK5-p35:13 nM, CDK4-CyclinD1:100 nM, CDK2-CyclinA:47 nM, CDK9-CyclinT:<10 nM, GSK-3β:89 nM

AT7519是一种与ATP竞争的CDK抑制剂，对CDK1的Ki值为38nM。除了对GSK3β（IC50=89nM）有活性外，AT7519对所有非CDK激酶均无活性。在多种人类肿瘤细胞系中表现出强大的抗增殖活性，IC50值从MCF-7的40nM到SW620的940nM不等，这与CDK1和CDK2的抑制一致。[1] AT7519在48小时内对多发性骨髓瘤（MM）细胞系产生剂量依赖性细胞毒性，IC50值从0.5μM到2μM不等，对MM.1S（0.5μM）和U266（0.5μM）细胞系最为敏感，对MM.1R（>2μM）耐药性较强。它不对周围血单个核细胞（PBMNC）产生细胞毒性。AT7519能部分克服IL6和IGF-1提供的增殖优势以及骨髓基质细胞（BMSCs）的保护作用。AT7519快速促使RNA聚合酶II CTD在丝氨酸2和丝氨酸5位点的去磷酸化，导致转录抑制，部分促成了对MM细胞的AT7519诱导的细胞毒性。通过下调GSK-3β磷酸化促进GSK-3β的激活，也为AT7519诱导的凋亡提供了帮助，这一过程与转录抑制无关。[2]

每日两次给予AT7519（9.1 mg/kg）可以导致HCT116和HT29结肠癌异种移植模型中，无论是早期还是晚期的皮下肿瘤退化。[1] AT7519治疗（15 mg/kg）抑制了人类MM异种移植小鼠模型中的肿瘤生长，并通过增加caspase 3激活，延长了小鼠的中位总生存时间。[2]

In vitro Kinase Assays: Kinase assays for CDK1, CDK2 and GSK3-β are all carried out in a radiometric filter binding format. Assays for CDK5 are in DELFIA format and for CDKs 4 and 6 in ELISA format. For CDKs 1 and 2, the relevant CDK and 0.12 μg/mL Histone H1 are incubated 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 different concentrations of AT7519 for 2 or 4 hours respectively. For GSK3-β, the relevant enzyme and 5 μM glycogen synthase peptide 2 along with 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) and different concentrations of AT7519 are incubated for 3 hours. Assay reactions are stopped by adding an excess of orthophosphoric acid and filtered using Millipore MAPH filter plates. The plates are then washed, scintillant added and radioactivity measured by scintillation counting on a Packard TopCount. For CDK5, CDK5/p35 and 1 μM of a biotinylated Histone H1 peptide (Biotin-PKTPKKAKKL) are incubated in 25 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.025% Brij-35, 0.1 mg/mL BSA, 1 mM DTT, 15 μM ATP and different concentrations of AT7519 for 30 minutes. Assay reactions are stopped using EDTA, transferred to Neutravidin-coated plates and phosphorylated peptide quantified by means of a rabbit phospho-cdk1 substrate polyclonal antibody and DELFIA europium-labelled anti-rabbit IgG secondary antibody using time-resolved fluorescence at λex=335 nM, λem=620 nM. For CDK 4 and 6 assays, plates are coated with GST- pRb769-921 and blocked with Superblock. CDK4 or 6 is incubated with 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 different concentrations of AT7519; the reaction is initiated by addition of ATP. After 30 minutes, reactions are stopped by the addition of 0.5 M EDTA pH 8.0.Plates are then washed and incubated for one hour with the primary antibody (anti- p-Rb Serine 780) diluted in Superblock followed by secondary antibody (alkaline phosphatase linked anti-rabbit) for a further hour. Plates are developed using the Attophos system and fluorescence read on a Spectramax Gemini plate reader at excitation 450 nm and emission 580 nm. In all cases, IC50 values are calculated from replicate curves, using GraphPad Prism software.

Cells are incubated with different concentrations of AT7519 for 24 or 48 hours at 37°C. Cell viability is assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrasodium bromide (MTT) dye absorbance. DNA synthesis is measured by tritiated thymidine uptake (3H-TdR). Apoptosis is assessed by using Annexin V/PI staining. The percentage of cells undergoing apoptosis is defined as the sum of early apoptosis (Annexin V-positive cells) and late apoptosis (Annexin V-positive and PI-positive cells(Only for Reference)