AZD2014

Reagent Authentication Certificate:

Chemical Probes Portal AZD2014.pdf

Protein target name:

MTOR

Mechanism of action:

ATP-competitive inhibitor

Recommended Concentration for use in cells:

10 nM - 2 uM

Probe Availability

MedChem Express (1009298-59-2)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Define other target class:

PIKK

Subclass:

Atypical

HUGO ID:

HGNC:3942

Entrez gene ID:

2475

UNIPROT ID:

P42345

Target alias:

SKS; FRAP; FRAP1; FRAP2; RAFT1; RAPT1

Probe Details

Chemical Probes Portal ID:

CPP106

Probe alias:

AZD-2014, Cc-551

PubChem CID:

25262792

ChEMBL ID:

CHEMBL2336325

SMILES string:

C[C@H]1COCCN1C2=NC(=NC3=C2C=CC(=N3)C4=CC(=CC=C4)C(=O)NC)N5CCOC[C@@H]5C

InChi Key:

JUSFANSTBFGBAF-IRXDYDNUSA-N

Orthogonal probe:

RapamycineCF309

PAINs evaluation:

PAINs free

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

0.0028 uM

Potency assay:

Inhibition of mTOR kinase was evaluated in a high-throughput assay using α screen capture complex technology (Perkin-Elmer) with a recombinant truncated FLAG-tagged mTOR (amino acids 1362–2549, expressed in HEK293 cells).

Off target activity

Off target protein and potency:

PI3Kalpha IC50=3.8 uMPI3K (other isoforms) IC50 > 30 uM

Potency assay (off target):

The activity of the lipid kinases, class I PI3Ks α, β, γ, δ was measured with the lipid PIP2 as substrate. AZD2014 was inactive against 200 additional kinases at 10 uM. AZD2014 is >1,000-fold selective for mTOR kinases over PI3K kinases (Ambit assay). In panels of 220 and 393 kinases, WNK2 was the only off-target hit with >50% inhibition and 10 uM, and minimal off-target binding was detected at 1 uM (Dudee and Millipore assays).

In cell validation

On target activity in cells

Potency in cells:

IC50

0.08 uM (pAKT)

Potency assay (cells):

Inhibition of substrate phosphorylation also yielded IC50 = 0.2 uM (pS6). Levels of phosphorylation were assessed using an Acumen laser scanning cytometer. In MDAMB468 cells, AZD2014 yielded an average IC50=210 nM for mTORC1 substrate ribosomal protein S6 and 78 nM for the MTORC2 substrate AKT. Comparable results were obtained in MCF7 cells.

Target engagement assay (cells):

Indirect, inhibition of protein substrate phosphorylation

In vivo validation

Organism:

CR-Han Wistar ratCD1 and SCID mouse

Dose:

4 uM/kg (rat)7.5-15 mg/kg (mouse)

Route of delivery:

OralIntravenous

Systemic clearance:

12.6 mL/min/kg (rat), 10.4 (mouse)

Fraction of probe bound to plasma protein:

0.91 (rat), 0.95 (mouse)

Organ of Interest

Organ (O):

MCF7 xenograft

Target engagement assay:

In xenografts, AZD2014 dose-dependently decrease protein-substrate phosphorylation (S6 and AKT).

Primary Reference

Reference (doi):

10.1016/j.bmcl.2013.01.019 10.1158/1535-7163.MCT-15-0365

Reference (PMID):

23375793 26358751

External Resources

Available data:

GDSC1 GDSC2

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells and in model organisms.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

Selectivity over 200 typical protein kinases and PI3Ks is >1000X. However, selectivity over atypical lipid-like protein kinases (e.g., ATM, ATR, DNA-PK) does not appear to have been disclosed, and this leaves outstanding selectivity concerns. In terms of the probe's use in cell-based assays, AZD8055 - a close analog - may be preferred, with potencies 5-10X greater than AZD2014. WYE-125132 may also be a preferable cell-based probe, with apparent superior cell-based potency over AZD2014, and reported >5000X selectivity over PI3Ks and some representative atypical protein kinases (ATR, hSMG1). With high bioavailability and low clearance in mice, AZD2014 is appropriate for human tumor xenograft models in mice; however, potency against murine mTor does not appear to have been disclosed. Suitability of the probe for use in a pure mouse model is unclear.

Jun 11 2016 - 4:37am

AZD2014

Probe Availability

Probe Information

Target Details

SKS; FRAP; FRAP1; FRAP2; RAFT1; RAPT1

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

External Resources

Supporting Organizations

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AZD2014

Probe Availability

Probe Information

Target Details

SKS; FRAP; FRAP1; FRAP2; RAFT1; RAPT1

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

External Resources

Supporting Organizations

Interested in supporting the portal?