Probe Criteria for Chemical Modulators vs PROTACs now available
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AT1
Protein target name:
BRD4
Mechanism of action:
DegraderPROTAC
Recommended Concentration for use in cells:
100-1000 nM
Probe Information
In vitro validation
Potency:
Kd
BRD4(BD1) 75 nM, (BD2) 45 nM
Potency assay:
Isothermal calorimetry (KD).
Structure-activity relationship:
SAR available as described in Gadd et al. Nat Chem Biol. doi:10.1038/nchembio.2329
AT2 (butyl), AT3 (PEG2), AT4 (poly-propyleneglycol-2), AT5 (mixed PEG-alkyl chain linker) and AT6 (PEG3) were all evaluated for their selectivity of intracellular degradation against BRD4 vs BRD2 vs BRD3.
AT1 has a linear hexyl chain linker
Potency (off target):
IC50
Probe Selectivity in Vitro:
Kinase off-target screening results. AT1 was screened at 1 μM concentration against a panel of 50 kinases (Dundee MRC-PPU Express Screen, http://www.kinase-screen.mrc.ac.uk/services/express- screen). The remaining kinase activity was recorded at the end of the assay. The data is reported as average % activity remaining of assay duplicates for each kinase tested (± standard deviation in brackets), ranked from highest to lowest. Plate Barcode AT1 D6769PM 1uM % activity (s.d.) TrkA 134 0
Src 125 19
SmMLCK 123 19
IGF-1R 123 7
HER4 118 3
MKK1 117 2
LKB1 113 5
Lck 113 12
GSK3b 112 14
RIPK2 109 1
MARK3 108 5
CK2 108 13
VEG-FR 107 8
PAK4 107 9
CAMK1 106 14
PRK2 106 3
SYK 105 4
BTK 105 4
PLK1 105 2
NEK6 105 12
EF2K 104 2
ROCK 2 104 2
JNK1 104 2
MST2 103 2
JAK3 103 3
TAK1 103 4
PKA 103 5
SGK1 103 1
PKD1 103 10
RSK1 102 1
CHK2 101 2
PKCa 101 9
EPH-A2 100 13
CK1δ 100 6
p38a MAPK 100 8
AMPK (hum) 99 2
PDK1 99 9
MLK3 99 9
HIPK2 99 0
MSK1 99 6
DYRK1A 97 1
PKBa 97 11
S6K1 95 7
TBK1 95 19
SRPK1 90 2
IRAK4 87 11
CAMKKb 87 7
PIM1 85 7
Aurora B 82 4
TTK 78 3
In cell validation
Potency in cells:
DC50
100 nM
Potency assay (cells):
Western blot (DC50 BRd4 = 100 nM) DC50 refers to the half maximal degradation concentration.
AT2 (butyl), AT3 (PEG2), AT4 (poly-propyleneglycol-2), AT5 (mixed PEG-alkyl chain linker) and AT6 (PEG3) were all evaluated for their selectivity of intracellular degradation against BRD4 vs BRD2 vs BRD3.
AT1 has a linear hexyl chain linker
Target engagement assay (cells):
n/a same mechanism as MZ1
Potency in cells, off target:
DC50
Off target protein and potency (cells):
Brd2, Brd3
Potency assay, off target (cells):
Western blot (DC50 BRd2 and BRd3 > 3 microM) DC50 refers to the half maximal degradation concentration.
Probe Selectivity in Cell:
Unbiased and quantitative isobaric tagging (TMT) mass spectrometry proteomics confirmed Brd4 as the sole protein markedly depleted (to ~40%) upon treatment with AT1 amongst the 5,674 detected proteins that passed filtering criteria (Fig. 4f and Supplementary Data Set 1 in GAdd et al.).
No effect on protein levels of Brd2 and Brd3 was observed with AT1.
Toxicity
Cytoxicity assay:
Yes
Notes on cytotoxicity:
AT1 showed cytotoxicity pEC50 of 5.9 (EC50 just above 1 uM) in BET sensitive AML cell line Mv4;11 in cell-titer glo assay