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Assay Detail

CHEMBL3888800

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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 ↗
320
Total Activities
305
Compounds Tested
1
Activity Types
0
Assay Parameters

Assay Information

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

Target

Serine/threonine-protein kinase pim-2 (CHEMBL4523) ↗
Type SINGLE PROTEIN
Organism Homo sapiens

Publication

Amides as pim inhibitors
(2016)

Activity Statistics

Type Count Avg pChEMBL Best pChEMBL
IC50 320 8.47 10.92

Compounds Tested

Compound Name Phase Activities Best pChEMBL
CHEMBL3940091 — — 1 10.92
CHEMBL3902148 — — 1 10.92
CHEMBL3909351 — — 1 10.70
CHEMBL3949102 — — 1 10.45
CHEMBL3903686 — — 1 10.45
CHEMBL3918907 — — 1 10.42
CHEMBL3895124 — — 1 10.42
CHEMBL3949318 — — 1 10.32
CHEMBL3949175 — — 1 10.22
CHEMBL3896195 — — 1 10.18
CHEMBL3960373 — — 1 10.13
CHEMBL3919725 — — 1 10.12
CHEMBL3933056 — — 1 10.11
CHEMBL3909217 — — 1 10.11
CHEMBL3941050 — — 1 10.00
CHEMBL3958862 — — 1 10.00
CHEMBL3918273 — — 1 10.00
CHEMBL3944421 — — 1 10.00
CHEMBL3901766 — — 1 10.00
CHEMBL3979182 — — 1 9.96
CHEMBL3919616 — — 1 9.96
CHEMBL3968854 — — 1 9.89
CHEMBL3957722 — — 1 9.87
CHEMBL3910446 — — 1 9.80
CHEMBL3890727 — — 1 9.79
CHEMBL3890346 — — 1 9.77
CHEMBL3942145 — — 1 9.75
CHEMBL3903402 — — 1 9.74
CHEMBL3924828 — — 1 9.72
CHEMBL3914535 — — 1 9.71

Activity Data

Compound Name Type Rel. Value Units pChEMBL
CHEMBL3940091 — IC50 = 0.012 nM 10.92
CHEMBL3902148 — IC50 = 0.012 nM 10.92
CHEMBL3909351 — IC50 = 0.02 nM 10.70
CHEMBL3949102 — IC50 = 0.0354 nM 10.45
CHEMBL3903686 — IC50 = 0.0358 nM 10.45
CHEMBL3895124 — IC50 = 0.0378 nM 10.42
CHEMBL3918907 — IC50 = 0.0378 nM 10.42
CHEMBL3949318 — IC50 = 0.0484 nM 10.32
CHEMBL3949175 — IC50 = 0.06 nM 10.22
CHEMBL3896195 — IC50 = 0.0667 nM 10.18
CHEMBL3960373 — IC50 = 0.0741 nM 10.13
CHEMBL3919725 — IC50 = 0.076 nM 10.12
CHEMBL3909217 — IC50 = 0.077 nM 10.11
CHEMBL3933056 — IC50 = 0.0783 nM 10.11
CHEMBL3941050 — IC50 = 0.1 nM 10.00
CHEMBL3901766 — IC50 = 0.1 nM 10.00
CHEMBL3958862 — IC50 = 0.1 nM 10.00
CHEMBL3944421 — IC50 = 0.1 nM 10.00
CHEMBL3918273 — IC50 = 0.1 nM 10.00
CHEMBL3979182 — IC50 = 0.109 nM 9.96
CHEMBL3919616 — IC50 = 0.11 nM 9.96
CHEMBL3968854 — IC50 = 0.13 nM 9.89
CHEMBL3957722 — IC50 = 0.136 nM 9.87
CHEMBL3910446 — IC50 = 0.16 nM 9.80
CHEMBL3890727 — IC50 = 0.162 nM 9.79
CHEMBL3890346 — IC50 = 0.169 nM 9.77
CHEMBL3942145 — IC50 = 0.177 nM 9.75
CHEMBL3903402 — IC50 = 0.183 nM 9.74
CHEMBL3924828 — IC50 = 0.19 nM 9.72
CHEMBL3897988 — IC50 = 0.193 nM 9.71
CHEMBL3914535 — IC50 = 0.195 nM 9.71
CHEMBL3947143 — IC50 = 0.193 nM 9.71
CHEMBL3979954 — IC50 = 0.201 nM 9.70
CHEMBL3911461 — IC50 = 0.2 nM 9.70
CHEMBL3896782 — IC50 = 0.2 nM 9.70
CHEMBL3940707 — IC50 = 0.201 nM 9.70
CHEMBL3984961 — IC50 = 0.2 nM 9.70
CHEMBL3905144 — IC50 = 0.212 nM 9.67
CHEMBL3919362 — IC50 = 0.218 nM 9.66
CHEMBL3896124 — IC50 = 0.223 nM 9.65
CHEMBL3924143 — IC50 = 0.242 nM 9.62
CHEMBL3934565 — IC50 = 0.24 nM 9.62
CHEMBL3937161 — IC50 = 0.246 nM 9.61
CHEMBL3913446 — IC50 = 0.267 nM 9.57
CHEMBL3927716 — IC50 = 0.272 nM 9.56
CHEMBL3919994 — IC50 = 0.278 nM 9.56
CHEMBL3897606 — IC50 = 0.275 nM 9.56
CHEMBL3903837 — IC50 = 0.297 nM 9.53
CHEMBL3943301 — IC50 = 0.305 nM 9.52
CHEMBL3966363 — IC50 = 0.301 nM 9.52