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Assay Detail

CHEMBL5731329

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Binding
ThermoFluor Assay: For the RORγt construct used in the ThermoFluor assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor assay is shown as SEQ ID NO:4. ThermoFluor experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
ChEMBL ↗
261
Total Activities
85
Compounds Tested
3
Activity Types
0
Assay Parameters

Assay Information

Assay Type Binding
Organism Homo sapiens
Confidence 9 — Direct single protein target
Curated By Autocuration

Target

Nuclear receptor ROR-gamma (CHEMBL1741186) ↗
Type SINGLE PROTEIN
Organism Homo sapiens

Publication

Thiazoles as modulators of RORγt
(2018)

Activity Statistics

Type Count Avg pChEMBL Best pChEMBL
Kd 87 9.14 10.72
kon 87 - -
k_off 87 - -

Compounds Tested

Compound Name Phase Activities Best pChEMBL
CHEMBL5821593 — — 3 10.72
CHEMBL5766445 — — 3 10.70
CHEMBL5848238 — — 3 10.70
CHEMBL5782981 — — 3 10.62
CHEMBL4642631 — — 3 10.60
CHEMBL5999201 — — 3 10.52
CHEMBL5812593 — — 3 10.44
CHEMBL4637144 — — 3 10.42
CHEMBL5948255 — — 3 10.40
CHEMBL6060307 — — 3 10.36
CHEMBL5870061 — — 3 10.35
CHEMBL5974922 — — 3 10.30
CHEMBL5772558 — — 3 10.30
CHEMBL6015906 — — 6 10.24
CHEMBL6046148 — — 3 10.22
CHEMBL5800104 — — 3 10.20
CHEMBL5745122 — — 3 10.15
CHEMBL5945490 — — 3 10.10
CHEMBL5849731 — — 3 10.08
CHEMBL6051987 — — 3 9.92
CHEMBL5841879 — — 3 9.92
CHEMBL5906971 — — 3 9.92
CHEMBL5893314 — — 3 9.92
CHEMBL5853965 — — 3 9.85
CHEMBL5835072 — — 3 9.82
CHEMBL5755744 — — 3 9.77
CHEMBL6028416 — — 3 9.70
CHEMBL6057262 — — 3 9.70
CHEMBL5976236 — — 3 9.68
CHEMBL5828299 — — 3 9.64

Activity Data

Compound Name Type Rel. Value Units pChEMBL
CHEMBL5821593 — Kd = 0.019 nM 10.72
CHEMBL5848238 — Kd = 0.02 nM 10.70
CHEMBL5766445 — Kd = 0.02 nM 10.70
CHEMBL5782981 — Kd = 0.024 nM 10.62
CHEMBL4642631 — Kd = 0.025 nM 10.60
CHEMBL5999201 — Kd = 0.03 nM 10.52
CHEMBL5812593 — Kd = 0.036 nM 10.44
CHEMBL4637144 — Kd = 0.038 nM 10.42
CHEMBL5948255 — Kd = 0.04 nM 10.40
CHEMBL6060307 — Kd = 0.044 nM 10.36
CHEMBL5870061 — Kd = 0.045 nM 10.35
CHEMBL5772558 — Kd = 0.05 nM 10.30
CHEMBL5974922 — Kd = 0.05 nM 10.30
CHEMBL6015906 — Kd = 0.058 nM 10.24
CHEMBL6046148 — Kd = 0.06 nM 10.22
CHEMBL5800104 — Kd = 0.063 nM 10.20
CHEMBL5745122 — Kd = 0.07 nM 10.15
CHEMBL5945490 — Kd = 0.08 nM 10.10
CHEMBL5849731 — Kd = 0.083 nM 10.08
CHEMBL5893314 — Kd = 0.12 nM 9.92
CHEMBL6051987 — Kd = 0.12 nM 9.92
CHEMBL5841879 — Kd = 0.12 nM 9.92
CHEMBL5906971 — Kd = 0.12 nM 9.92
CHEMBL5853965 — Kd = 0.14 nM 9.85
CHEMBL5835072 — Kd = 0.15 nM 9.82
CHEMBL5755744 — Kd = 0.17 nM 9.77
CHEMBL6057262 — Kd = 0.2 nM 9.70
CHEMBL6028416 — Kd = 0.2 nM 9.70
CHEMBL5976236 — Kd = 0.21 nM 9.68
CHEMBL5828299 — Kd = 0.23 nM 9.64
CHEMBL5872042 — Kd = 0.25 nM 9.60
CHEMBL6015906 — Kd = 0.27 nM 9.57
CHEMBL5818006 — Kd = 0.27 nM 9.57
CHEMBL5908815 — Kd = 0.28 nM 9.55
CHEMBL5786068 — Kd = 0.29 nM 9.54
CHEMBL5897038 — Kd = 0.32 nM 9.49
CHEMBL5773278 — Kd = 0.33 nM 9.48
CHEMBL6033015 — Kd = 0.33 nM 9.48
CHEMBL5782103 — Kd = 0.33 nM 9.48
CHEMBL5849047 — Kd = 0.35 nM 9.46
CHEMBL4642450 — Kd = 0.35 nM 9.46
CHEMBL5971406 — Kd = 0.36 nM 9.44
CHEMBL5890321 — Kd = 0.39 nM 9.41
CHEMBL4645288 — Kd = 0.46 nM 9.34
CHEMBL5812569 — Kd = 0.47 nM 9.33
CHEMBL5904989 — Kd = 0.55 nM 9.26
CHEMBL5993790 — Kd = 0.62 nM 9.21
CHEMBL5903843 — Kd = 0.62 nM 9.21
CHEMBL6010818 — Kd = 0.71 nM 9.15
CHEMBL4648607 — Kd = 0.82 nM 9.09