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Assay Detail

CHEMBL5738267

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Binding
CDK6/Cyclin D1 Mobility Shift Assay: The purpose of the CDK6/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK6/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). 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. Typical reaction solutions contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 3 nM (active sites) CDK6/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK6/CyclinD1 (LJIC-2003A2/1865) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
ChEMBL ↗
597
Total Activities
192
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

CDK6/cyclin D1 (CHEMBL2111455) ↗
Type PROTEIN COMPLEX
Organism Homo sapiens

Publication

CDK2/4/6 inhibitors
(2022)

Activity Statistics

Type Count Avg pChEMBL Best pChEMBL
Ki 199 8.73 10.22
kon 199 - -
k_off 199 - -

Compounds Tested

Compound Name Phase Activities Best pChEMBL
CHEMBL5817327 — — 3 10.22
CHEMBL4446357 EBVACICLIB 2.0 3 10.10
CHEMBL5994083 — — 3 10.10
CHEMBL5833205 — — 6 9.96
CHEMBL5774115 — — 3 9.92
CHEMBL5281860 — — 3 9.89
CHEMBL5281770 — — 3 9.89
CHEMBL6047972 — — 3 9.89
CHEMBL5764489 — — 3 9.85
CHEMBL5866256 — — 3 9.85
CHEMBL5789495 — — 3 9.82
CHEMBL5857212 — — 3 9.77
CHEMBL4745424 — — 3 9.74
CHEMBL5942490 — — 3 9.74
CHEMBL5269912 — — 3 9.72
CHEMBL5926192 — — 3 9.68
CHEMBL5790162 — — 3 9.64
CHEMBL5964436 — — 3 9.64
CHEMBL5847372 — — 3 9.62
CHEMBL5271753 — — 3 9.60
CHEMBL5278622 — — 3 9.59
CHEMBL5793277 — — 3 9.59
CHEMBL5752026 — — 3 9.57
CHEMBL6042439 — — 3 9.55
CHEMBL5963572 — — 3 9.54
CHEMBL4796501 — — 3 9.51
CHEMBL5272948 — — 3 9.48
CHEMBL6005561 — — 3 9.48
CHEMBL5906572 — — 3 9.47
CHEMBL5916238 — — 3 9.44

Activity Data

Compound Name Type Rel. Value Units pChEMBL
CHEMBL5817327 — Ki = 0.06 nM 10.22
CHEMBL5994083 — Ki = 0.08 nM 10.10
CHEMBL4446357 EBVACICLIB Ki = 0.08 nM 10.10
CHEMBL5833205 — Ki = 0.11 nM 9.96
CHEMBL5774115 — Ki = 0.12 nM 9.92
CHEMBL6047972 — Ki = 0.13 nM 9.89
CHEMBL5281860 — Ki = 0.13 nM 9.89
CHEMBL5281770 — Ki = 0.13 nM 9.89
CHEMBL5764489 — Ki = 0.14 nM 9.85
CHEMBL5866256 — Ki = 0.14 nM 9.85
CHEMBL5789495 — Ki = 0.15 nM 9.82
CHEMBL5857212 — Ki = 0.17 nM 9.77
CHEMBL4745424 — Ki = 0.18 nM 9.74
CHEMBL5942490 — Ki = 0.18 nM 9.74
CHEMBL5833205 — Ki = 0.19 nM 9.72
CHEMBL5269912 — Ki = 0.19 nM 9.72
CHEMBL5926192 — Ki = 0.21 nM 9.68
CHEMBL5790162 — Ki = 0.23 nM 9.64
CHEMBL5964436 — Ki = 0.23 nM 9.64
CHEMBL5847372 — Ki = 0.24 nM 9.62
CHEMBL5271753 — Ki = 0.25 nM 9.60
CHEMBL5793277 — Ki = 0.26 nM 9.59
CHEMBL5278622 — Ki = 0.26 nM 9.59
CHEMBL5752026 — Ki = 0.27 nM 9.57
CHEMBL6042439 — Ki = 0.28 nM 9.55
CHEMBL5963572 — Ki = 0.29 nM 9.54
CHEMBL4796501 — Ki = 0.31 nM 9.51
CHEMBL5272948 — Ki = 0.33 nM 9.48
CHEMBL6005561 — Ki = 0.33 nM 9.48
CHEMBL5906572 — Ki = 0.34 nM 9.47
CHEMBL5940340 — Ki = 0.36 nM 9.44
CHEMBL5916238 — Ki = 0.36 nM 9.44
CHEMBL5864655 — Ki = 0.37 nM 9.43
CHEMBL5868351 — Ki = 0.37 nM 9.43
CHEMBL5844501 — Ki = 0.38 nM 9.42
CHEMBL5871813 — Ki = 0.42 nM 9.38
CHEMBL5831969 — Ki = 0.42 nM 9.38
CHEMBL5870144 — Ki = 0.43 nM 9.37
CHEMBL5838779 — Ki = 0.43 nM 9.37
CHEMBL6065332 — Ki = 0.44 nM 9.36
CHEMBL5850433 — Ki = 0.45 nM 9.35
CHEMBL5940313 — Ki = 0.49 nM 9.31
CHEMBL5833936 — Ki = 0.53 nM 9.28
CHEMBL5816880 — Ki = 0.53 nM 9.28
CHEMBL5792057 — Ki = 0.54 nM 9.27
CHEMBL5962341 — Ki = 0.55 nM 9.26
CHEMBL5866017 — Ki = 0.58 nM 9.24
CHEMBL5760712 — Ki = 0.62 nM 9.21
CHEMBL6038741 — Ki = 0.63 nM 9.20
CHEMBL5971072 — Ki = 0.66 nM 9.18