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Assay Detail

CHEMBL3888799

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Binding
Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
ChEMBL ↗
322
Total Activities
306
Compounds Tested
1
Activity Types
0
Assay Parameters

Assay Information

Assay Type Binding
Confidence 8 — Homologous single protein target
Curated By Autocuration

Target

Serine/threonine-protein kinase pim-1 (CHEMBL2147) ↗
Type SINGLE PROTEIN
Organism Homo sapiens

Publication

Amides as pim inhibitors
(2016)

Activity Statistics

Type Count Avg pChEMBL Best pChEMBL
IC50 322 8.56 10.92

Compounds Tested

Compound Name Phase Activities Best pChEMBL
CHEMBL3902148 — — 1 10.92
CHEMBL3895124 — — 1 10.89
CHEMBL3918907 — — 2 10.55
CHEMBL3960373 — — 1 10.54
CHEMBL3949102 — — 1 10.53
CHEMBL3933056 — — 1 10.47
CHEMBL3949318 — — 1 10.42
CHEMBL3909351 — — 1 10.40
CHEMBL3949175 — — 1 10.34
CHEMBL3919725 — — 1 10.23
CHEMBL3903686 — — 1 10.22
CHEMBL3941050 — — 1 10.19
CHEMBL3940091 — — 1 10.15
CHEMBL3944421 — — 1 10.15
CHEMBL3968854 — — 1 10.13
CHEMBL3909217 — — 1 10.09
CHEMBL3901766 — — 1 10.00
CHEMBL3934565 — — 1 10.00
CHEMBL3984961 — — 1 10.00
CHEMBL3958862 — — 1 10.00
CHEMBL3896782 — — 1 10.00
CHEMBL3979182 — — 1 9.94
CHEMBL3919616 — — 1 9.92
CHEMBL3896124 — — 1 9.86
CHEMBL3924828 — — 1 9.80
CHEMBL3890346 — — 1 9.75
CHEMBL3940707 — — 1 9.74
CHEMBL3962064 — — 1 9.74
CHEMBL3946152 — — 1 9.74
CHEMBL3903402 — — 1 9.73

Activity Data

Compound Name Type Rel. Value Units pChEMBL
CHEMBL3902148 — IC50 = 0.012 nM 10.92
CHEMBL3895124 — IC50 = 0.013 nM 10.89
CHEMBL3918907 — IC50 = 0.0282 nM 10.55
CHEMBL3960373 — IC50 = 0.029 nM 10.54
CHEMBL3949102 — IC50 = 0.0295 nM 10.53
CHEMBL3933056 — IC50 = 0.0339 nM 10.47
CHEMBL3949318 — IC50 = 0.0376 nM 10.42
CHEMBL3909351 — IC50 = 0.04 nM 10.40
CHEMBL3949175 — IC50 = 0.046 nM 10.34
CHEMBL3919725 — IC50 = 0.0586 nM 10.23
CHEMBL3903686 — IC50 = 0.0598 nM 10.22
CHEMBL3941050 — IC50 = 0.065 nM 10.19
CHEMBL3944421 — IC50 = 0.07 nM 10.15
CHEMBL3940091 — IC50 = 0.07 nM 10.15
CHEMBL3968854 — IC50 = 0.074 nM 10.13
CHEMBL3909217 — IC50 = 0.082 nM 10.09
CHEMBL3896782 — IC50 = 0.1 nM 10.00
CHEMBL3958862 — IC50 = 0.1 nM 10.00
CHEMBL3984961 — IC50 = 0.1 nM 10.00
CHEMBL3901766 — IC50 = 0.1 nM 10.00
CHEMBL3934565 — IC50 = 0.1 nM 10.00
CHEMBL3979182 — IC50 = 0.116 nM 9.94
CHEMBL3919616 — IC50 = 0.12 nM 9.92
CHEMBL3896124 — IC50 = 0.139 nM 9.86
CHEMBL3924828 — IC50 = 0.16 nM 9.80
CHEMBL3890346 — IC50 = 0.178 nM 9.75
CHEMBL3940707 — IC50 = 0.181 nM 9.74
CHEMBL3962064 — IC50 = 0.18 nM 9.74
CHEMBL3946152 — IC50 = 0.18 nM 9.74
CHEMBL3903402 — IC50 = 0.185 nM 9.73
CHEMBL3890727 — IC50 = 0.184 nM 9.73
CHEMBL3957722 — IC50 = 0.191 nM 9.72
CHEMBL3942145 — IC50 = 0.192 nM 9.72
CHEMBL3979954 — IC50 = 0.195 nM 9.71
CHEMBL3947143 — IC50 = 0.199 nM 9.70
CHEMBL3911461 — IC50 = 0.2 nM 9.70
CHEMBL3918273 — IC50 = 0.2 nM 9.70
CHEMBL3965643 — IC50 = 0.2 nM 9.70
CHEMBL3926782 — IC50 = 0.2 nM 9.70
CHEMBL3950704 — IC50 = 0.238 nM 9.62
CHEMBL3896195 — IC50 = 0.246 nM 9.61
CHEMBL3897988 — IC50 = 0.249 nM 9.60
CHEMBL3943763 — IC50 = 0.249 nM 9.60
CHEMBL3937161 — IC50 = 0.258 nM 9.59
CHEMBL3897606 — IC50 = 0.259 nM 9.59
CHEMBL3933719 — IC50 = 0.255 nM 9.59
CHEMBL3914535 — IC50 = 0.271 nM 9.57
CHEMBL3910446 — IC50 = 0.28 nM 9.55
CHEMBL3926620 — IC50 = 0.287 nM 9.54
CHEMBL3943301 — IC50 = 0.289 nM 9.54