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Assay Detail

CHEMBL5736835

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Binding
Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
ChEMBL ↗
1947
Total Activities
569
Compounds Tested
3
Activity Types
0
Assay Parameters

Assay Information

Assay Type Binding
Organism Homo sapiens
Confidence 7 — Homologous single protein target
Curated By Autocuration

Target

Cyclin-dependent kinase 2/cyclin E1 (CHEMBL1907605) ↗
Type PROTEIN COMPLEX
Organism Homo sapiens

Publication

CDK2 inhibitors
(2021)

Activity Statistics

Type Count Avg pChEMBL Best pChEMBL
Ki 649 9.14 10.40
kon 649 - -
k_off 649 - -

Compounds Tested

Compound Name Phase Activities Best pChEMBL
CHEMBL5899718 — — 3 10.40
CHEMBL5762472 — — 3 10.30
CHEMBL5857215 — — 3 10.22
CHEMBL5808436 — — 3 10.15
CHEMBL5975537 — — 6 10.15
CHEMBL5805501 — — 6 10.10
CHEMBL5876770 — — 3 10.10
CHEMBL5765623 — — 3 10.05
CHEMBL5813951 — — 6 10.05
CHEMBL5994701 — — 3 10.05
CHEMBL5893129 — — 3 10.00
CHEMBL4744945 — — 3 10.00
CHEMBL5883663 — — 6 10.00
CHEMBL5776785 — — 3 10.00
CHEMBL5804029 — — 3 10.00
CHEMBL6047683 — — 3 10.00
CHEMBL5909080 — — 6 10.00
CHEMBL5948920 — — 3 9.96
CHEMBL6022715 — — 3 9.96
CHEMBL5886881 — — 3 9.96
CHEMBL5828800 — — 3 9.96
CHEMBL5921041 — — 3 9.96
CHEMBL5756851 — — 3 9.92
CHEMBL4764153 — — 3 9.92
CHEMBL5170427 — — 3 9.89
CHEMBL5887015 — — 3 9.89
CHEMBL5987162 — — 3 9.89
CHEMBL5880964 — — 6 9.89
CHEMBL5772687 — — 6 9.89
CHEMBL5754961 — — 3 9.85

Activity Data

Compound Name Type Rel. Value Units pChEMBL
CHEMBL5899718 — Ki = 0.04 nM 10.40
CHEMBL5762472 — Ki = 0.05 nM 10.30
CHEMBL5857215 — Ki = 0.06 nM 10.22
CHEMBL5975537 — Ki = 0.07 nM 10.15
CHEMBL5808436 — Ki = 0.07 nM 10.15
CHEMBL5805501 — Ki = 0.08 nM 10.10
CHEMBL5975537 — Ki = 0.08 nM 10.10
CHEMBL5876770 — Ki = 0.08 nM 10.10
CHEMBL5805501 — Ki = 0.09 nM 10.05
CHEMBL5813951 — Ki = 0.09 nM 10.05
CHEMBL5765623 — Ki = 0.09 nM 10.05
CHEMBL5994701 — Ki = 0.09 nM 10.05
CHEMBL6047683 — Ki = 0.1 nM 10.00
CHEMBL5893129 — Ki = 0.1 nM 10.00
CHEMBL4744945 — Ki = 0.1 nM 10.00
CHEMBL5883663 — Ki = 0.1 nM 10.00
CHEMBL5909080 — Ki = 0.1 nM 10.00
CHEMBL5776785 — Ki = 0.1 nM 10.00
CHEMBL5804029 — Ki = 0.1 nM 10.00
CHEMBL5828800 — Ki = 0.11 nM 9.96
CHEMBL5886881 — Ki = 0.11 nM 9.96
CHEMBL6022715 — Ki = 0.11 nM 9.96
CHEMBL5921041 — Ki = 0.11 nM 9.96
CHEMBL5948920 — Ki = 0.11 nM 9.96
CHEMBL4764153 — Ki = 0.12 nM 9.92
CHEMBL5756851 — Ki = 0.12 nM 9.92
CHEMBL5887015 — Ki = 0.13 nM 9.89
CHEMBL5987162 — Ki = 0.13 nM 9.89
CHEMBL5880964 — Ki = 0.13 nM 9.89
CHEMBL5772687 — Ki = 0.13 nM 9.89
CHEMBL5170427 — Ki = 0.13 nM 9.89
CHEMBL5740073 — Ki = 0.14 nM 9.85
CHEMBL5754961 — Ki = 0.14 nM 9.85
CHEMBL6063713 — Ki = 0.14 nM 9.85
CHEMBL4758192 — Ki = 0.15 nM 9.82
CHEMBL5801175 — Ki = 0.15 nM 9.82
CHEMBL6007401 — Ki = 0.15 nM 9.82
CHEMBL5787735 — Ki = 0.15 nM 9.82
CHEMBL5968347 — Ki = 0.15 nM 9.82
CHEMBL5800918 — Ki = 0.15 nM 9.82
CHEMBL5828704 — Ki = 0.15 nM 9.82
CHEMBL5751251 — Ki = 0.15 nM 9.82
CHEMBL5842985 — Ki = 0.15 nM 9.82
CHEMBL5792819 — Ki = 0.15 nM 9.82
CHEMBL5864784 — Ki = 0.15 nM 9.82
CHEMBL5919074 — Ki = 0.15 nM 9.82
CHEMBL5818259 — Ki = 0.16 nM 9.80
CHEMBL5900076 — Ki = 0.16 nM 9.80
CHEMBL5866244 — Ki = 0.16 nM 9.80
CHEMBL5913529 — Ki = 0.16 nM 9.80