AT1

Protein target name:

Brd4

Mechanism of action:

Protelysis Targeting Chimeric Molecule (proteasome-mediated degradation)

Recommended Concentration for use in cells:

100-1000 nM

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Bromodomain

HUGO symbol:

HGNC:13575

Entrez gene ID:

23476

Target alias:

Transcriptional coregulator

Probe Details

Chemical Probes Portal ID:

CPP1827

Probe alias:

PROTAC

PubChem CID:

124201841

SMILES string:

O[C@@H]1C[C@@H](C(NCC2=CC=C(C3=C(C)N=CS3)C=C2)=O)N(C([C@@H](NC(C)=O)C(SCCCCCCNC(C[C@@H]4N=C(C5=CC=C(Cl)C=C5)C6=C(SC(C)=C6C)N7C4=NN=C7C)=O)(C)C)=O)C1

InChi Key:

SQNZDYHMCMIGGV-TZPPCSJFSA-N

Control compound:

cis-AT1

Orthogonal probe:

MZ1dBET1, ARV-825

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

10 mM

Potential reactivities:

not known

NMR:

1H-NMR (400 MHz, CD3OD, 25 °C) δ: 8.99 (s, 1H), 8. 43 (t, J = 5.9 Hz, 1 H exch.), 8.12 (d, J = 9.4 Hz, 1H exch.), 8.07 (s, 1H exch.), 7.47-7.40 (m, 8H), 4.93-4.91 (m, 1H), 4.69-4.65 (m, 1H), 4.58 (t, J = 8.3 Hz, 1H), 4.52-4.38 (m, 3H), 3.93-3.91 (m, 1H), 3.85 (dd, J = 10.8 Hz, J = 3.96 Hz, 1H), 3.45-3.39 (m, 1H), 3.27-3.16 (m, 3H), 2.72 (s, 3H), 2.26 (t, J = 7.0 Hz, 2H), 2.49 (s, 3H), 2.45 (s, 3H), 2.25-2.22 (m, 1H), 2.14-2.07 (m, 1H), 2.00 (s, 3H), 1.70 (s, 3H), 1.54-1.36 (m, 14H).
13C-NMR (101 MHz, CD3OD, 25 °C) δ: 174.4, 173.2, 172.6, 171.6, 166.7, 157.1, 153.4, 152.6, 148.5, 140.5, 138.4, 137.9, 133.9, 133.6, 132.3, 132.2, 131.6, 131.4, 130.6, 130.0, 129.7, 129.3, 71.0, 61.2, 58.1, 57.4, 55.2, 43.7, 40.5, 39.2, 38.7, 30.7, 30.5, 30.0, 29.3, 27.7, 26.2, 25.9, 22.5, 15.7, 14.6, 13.1, 11.7.

Storage Recommendations

Storage (dry powder):

dry powder -20

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

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

Off target activity

Potency (off target):

IC50

Selectivity against non-family targets:

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

On target activity in cells

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

Off target activity in cells

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.

Selectivity against nonfamily targets (cells):

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

Primary Reference

Reference (doi):

10.1038/nchembio.2329

Reference (PMID):

28288108

This probe is in the process of SAB review. Please continue to check back for new reviews and commentary.

SAB Rating

Rating for use in Cells

2 review(s)

Rating for use in Model Organisms

2 review(s)

SAB Members

SAB Comments

AT1 is a very good PROTAC ligand for targeted degradation of BRD4. Improved from previous PROTAC MZ1, AT1 has superior selectivity towards BRD4 in the BET family while retaining its potency (DC50 = 100nM). The paper has provided ample evidence detailing that selectivity is achieved via ligand-induced protein-protein interaction (PPI) between BRD4 and VHL. AT1 is thus structurally optimized from MZ1 to facilitate PPI. However, the ITC measurements do not fully explained AT1’s superior selectivity. More data is also needed for the probe’s use in organisms (e.g. PK and toxicity studies).

AT1

Probe Information

Target Details

Transcriptional coregulator

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

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AT1

Probe Information

Target Details

Transcriptional coregulator

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?