Skip to main content
Free research Free research Free assay review with research home and workspace preview
Quick search ChEMBL 36
Assay Detail

CHEMBL3889266

Review assay metadata, readout intent, target linkage, and publication context from the same page.

Binding
LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 uM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 uL) was dispensed, followed by the addition of 7.5 uL of 1x kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/uL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 uL 1x kinase buffer containing 8 uM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ ID NO: 1), and 100 uM ATP. The final reaction in each well of 10 uL consists of 100 pM hBTK, 2 uM biotin-A5-peptide, and 25 uM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1x quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
ChEMBL ↗
221
Total Activities
220
Compounds Tested
1
Activity Types
0
Assay Parameters

Assay Information

Assay Type Binding
Organism Homo sapiens
Cell Type Sf9
Confidence 8 — Homologous single protein target
Curated By Autocuration

Target

Tyrosine-protein kinase BTK (CHEMBL5251) ↗
Type SINGLE PROTEIN
Organism Homo sapiens

Publication

Substituted benzamides and substituted pyridinecarboxamides as Btk inhibitors
(2016)

Activity Statistics

Type Count Avg pChEMBL Best pChEMBL
IC50 221 8.79 10.24

Compounds Tested

Compound Name Phase Activities Best pChEMBL
CHEMBL4113670 — — 1 10.24
CHEMBL4113445 — — 1 10.12
CHEMBL4109744 — — 1 10.09
CHEMBL4109282 — — 1 10.08
CHEMBL3938520 — — 1 10.07
CHEMBL4109792 — — 1 10.04
CHEMBL4108545 — — 1 10.02
CHEMBL3924979 — — 1 10.02
CHEMBL4106978 — — 1 10.00
CHEMBL4108563 — — 1 9.96
CHEMBL4110644 — — 1 9.96
CHEMBL4112025 — — 1 9.92
CHEMBL4108825 — — 1 9.92
CHEMBL4108593 — — 1 9.89
CHEMBL4107482 — — 1 9.89
CHEMBL4108295 — — 1 9.89
CHEMBL4108579 — — 1 9.89
CHEMBL4108687 — — 1 9.89
CHEMBL4109294 — — 1 9.89
CHEMBL3942777 — — 1 9.85
CHEMBL3916919 — — 1 9.85
CHEMBL4111650 — — 1 9.80
CHEMBL4110388 — — 1 9.80
CHEMBL4106573 — — 1 9.80
CHEMBL3922148 — — 1 9.77
CHEMBL4110653 — — 1 9.77
CHEMBL4111363 — — 1 9.77
CHEMBL4113354 — — 1 9.74
CHEMBL4113263 — — 1 9.74
CHEMBL4109144 — — 1 9.72

Activity Data

Compound Name Type Rel. Value Units pChEMBL
CHEMBL4113670 — IC50 = 0.058 nM 10.24
CHEMBL4113445 — IC50 = 0.075 nM 10.12
CHEMBL4109744 — IC50 = 0.081 nM 10.09
CHEMBL4109282 — IC50 = 0.083 nM 10.08
CHEMBL3938520 — IC50 = 0.085 nM 10.07
CHEMBL4109792 — IC50 = 0.092 nM 10.04
CHEMBL3924979 — IC50 = 0.095 nM 10.02
CHEMBL4108545 — IC50 = 0.096 nM 10.02
CHEMBL4106978 — IC50 = 0.1 nM 10.00
CHEMBL4110644 — IC50 = 0.11 nM 9.96
CHEMBL4108563 — IC50 = 0.11 nM 9.96
CHEMBL4112025 — IC50 = 0.12 nM 9.92
CHEMBL4108825 — IC50 = 0.12 nM 9.92
CHEMBL4108593 — IC50 = 0.13 nM 9.89
CHEMBL4107482 — IC50 = 0.13 nM 9.89
CHEMBL4109294 — IC50 = 0.13 nM 9.89
CHEMBL4108295 — IC50 = 0.13 nM 9.89
CHEMBL4108687 — IC50 = 0.13 nM 9.89
CHEMBL4108579 — IC50 = 0.13 nM 9.89
CHEMBL3942777 — IC50 = 0.14 nM 9.85
CHEMBL3916919 — IC50 = 0.14 nM 9.85
CHEMBL4110388 — IC50 = 0.16 nM 9.80
CHEMBL4111650 — IC50 = 0.16 nM 9.80
CHEMBL4106573 — IC50 = 0.16 nM 9.80
CHEMBL3922148 — IC50 = 0.17 nM 9.77
CHEMBL4110653 — IC50 = 0.17 nM 9.77
CHEMBL4111363 — IC50 = 0.17 nM 9.77
CHEMBL4113354 — IC50 = 0.18 nM 9.74
CHEMBL4113263 — IC50 = 0.18 nM 9.74
CHEMBL4107467 — IC50 = 0.19 nM 9.72
CHEMBL4109144 — IC50 = 0.19 nM 9.72
CHEMBL4114929 — IC50 = 0.19 nM 9.72
CHEMBL4114097 — IC50 = 0.2 nM 9.70
CHEMBL4112223 — IC50 = 0.2 nM 9.70
CHEMBL4111521 — IC50 = 0.21 nM 9.68
CHEMBL4109557 — IC50 = 0.21 nM 9.68
CHEMBL4112274 — IC50 = 0.21 nM 9.68
CHEMBL4112201 — IC50 = 0.21 nM 9.68
CHEMBL4111121 — IC50 = 0.22 nM 9.66
CHEMBL4114929 — IC50 = 0.22 nM 9.66
CHEMBL4111645 — IC50 = 0.22 nM 9.66
CHEMBL4107715 — IC50 = 0.22 nM 9.66
CHEMBL4109377 — IC50 = 0.23 nM 9.64
CHEMBL4110406 — IC50 = 0.23 nM 9.64
CHEMBL4112085 — IC50 = 0.24 nM 9.62
CHEMBL4114094 — IC50 = 0.26 nM 9.59
CHEMBL4113994 — IC50 = 0.26 nM 9.59
CHEMBL4111825 — IC50 = 0.27 nM 9.57
CHEMBL4109533 — IC50 = 0.27 nM 9.57
CHEMBL4112426 — IC50 = 0.28 nM 9.55