JAK3i

Protein target name:

JAK3

Mechanism of action:

Irreversible inhibitor

Recommended Concentration for use in cells:

15-250 nM

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

HUGO ID:

HGNC:6193

Entrez gene ID:

3718

UNIPROT ID:

P52333

Target alias:

JAK-3_HUMAN, JAKL, L-JAK, LJAK, JAK3, Janus kinase 3

Probe Details

Chemical Probes Portal ID:

CPP657

PubChem CID:

118705186

SMILES string:

CCOC(=O)C1=CNC2=NC=NC(=C12)C3=C(C=CC(=C3)NC(=O)C=C)F

InChi Key:

KNLROUBMVYVLGX-UHFFFAOYSA-N

Control compound:

Compound 2 from Smith et al. 2016 (propionamide of JAK3i)

Orthogonal probe:

TofacitinibFM-381

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

>10 mM

NMR:

1H NMR (400 MHz, DMSO): 13.04 (br s, 1H), 10.33 (s, 1H), 8.97 (s, 1H), 8.33 (s, 1H), 8.06 (dd, J = 2.7, 6.6 Hz, 1H), 7.83 (ddd, J = 2.7, 4.7, 9.0 Hz, 1H), 7.23 (dd, J = 9.0, 9.0 Hz, 1H), 6.44 (dd, J = 10.2, 16.8 Hz, 1H), 6.28 (dd, J = 2.0, 16.8 Hz, 1H), 5.77 (dd, J = 2.0, 10.2 Hz, 1H), 4.01 (br s, 1H), 3.88 (br s, 1H), 0.94 (t, J = 7.4 Hz, 3H).

13C NMR (100 MHz, DMSO): 163.1, 162.8, 156.1 (J = 243 Hz), 153.6, 153.0, 151.6, 135.0 (J = 2.3 Hz), 134.6, 131.7, 127.5 (J = 15.3 Hz), 127.0, 121.8 (J = 8.4 Hz), 121.5 (J = 3.1 Hz), 115.2 (J = 23.7 Hz), 113.8, 107.7 (J = 3.1 Hz), 59.5, 13.7. ESI-MS: 355.4 (M+H+).

Storage Recommendations

Storage (dry powder):

Room temperature

Storage (solution):

-20 C, stable for >2 years.

Storage sensitivities:

Not sensitive to freeze-thaw.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

0.43 nM

Potency assay:

JAK3 in vitro kinase assay with JAK3 kinase domain (Invitrogen), 100 uM ATP, 30 minute pre-incubation and 30 minute incubation with substrate (JAK3Tide) + 32P ATP.

Off target activity

Potency (off target):

IC50

Off target protein and potency:

JAK1 1,520 nMJAK2 8,680 nMTYK2 >10 uMBTK 363 nMITK 4,630 nMEGFR >10 uM

Potency assay (off target):

In vitro kinase assays with purified kinase domains, monitored by 32P-ATP. JAK1, JAK2, TYK2 were assayed with 100 uM ATP. BTK, ITK and EGFR were assayed with 1 mM ATP (IC50 against JAK3 with 1 mM ATP = 7.43 nM). In biochemical assays, JAK3i is >3,000-fold selective for JAK3 over JAK1, JAK2 and TYK2. JAK3i was also highly selective for JAK3 over TEC family kinases, displaying 1,300-fold selectivity over EGFR, 600-fold over ITK and 50-fold over BTK.

Probe Selectivity in Vitro:

Not available

In cell validation

On target activity in cells

Potency in cells:

IC50

3.7 nM

Potency assay (cells):

IL-2-induced proliferation of CD4+ T-cell blasts from mouse. Briefly, CD4 T-cell blasts were stimulated with 50 units/mL of rhIL-2, incubated for 24 hours, and then proliferation was assessed by 24-hour tritiated thymidine uptake. Multiple assays downstream of IL-2 were performed with some variation in IC50: 47 nM for formation of pSTAT5 (FACS), 3.1 nM for upregulation of IL-2Ralpha (CD25) at 24-hours post-IL-2 stimulation, 6.8 nM for IFN-gamma production 24-hours post-IL-2 stimulation.

Target engagement assay (cells):

To validate target engagement and on-target effects in cells, a JAK3i-resistant JAK3 mutant (Cys905Ser) was overexpressed in primary murine T cells. The IC50 in cells overexpressing the C905S mutant shifts to >4 uM from <4 nM in untransduced cells (1,000-fold potency shift), providing strong evidence that covalent binding to JAK3 is required for cellular activity. Similar rescues were observed in assays of STAT5 phosphorylation and CD25 upregulation with the mutant protein.

Off target activity in cells

Potency in cells, off target:

IC50

Off target protein and potency (cells):

BTK >10 uMJAK1/JAK2 >10 uMITK/RLK 1,172 nM

Potency assay, off target (cells):

BTK: BCR-stimulated CD69 production in primary mouse splenic B cells (ibrutinib, control compound for BTK, IC50 <1 nM). JAK1/JAK2: IFNy-stimulated pSTAT1 (measured by FACS) in CD4+ T-cell blasts (tofacitinib, control compound for JAK1, JAK2, TYK2, IC50 63.01 nM) ITK/RLK: TCR-stimulated IL-2 production in primary murine CD4 T cells, determined by ELISA (PF-06465469, control compound for ITK, IC50 4.4 nM)

Probe Selectivity in Cell:

n/a

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

No affect on Jurkat T cells up to 10 uM (Alamar Blue assay)

In vivo validation

Organism:

mouse

Dose:

40 mg/kg

Route of delivery:

Intraperitoneal

Plasma half life:

~50 minutes

Organ of Interest

Organ (O):

SpleenLymph node

Target engagement assay:

Overexpression of the drug resistant Cys to Ser mutant provides evidence that JAK3 binding is required for in vivo activity. Cells overexpressing C905S JAK3 were resistant to JAK3i in in vivo proliferation assays, while cells overexpressing wild-type JAK3 in the same mouse were sensitive to JAK3i. Cells in these assays came from the spleen.

Primary Reference

Reference (doi):

10.1038/nchembio.2056

Reference (PMID):

27018889

Endorsed probe

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

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

Broad kinase selectivity of this probe needs to be determined before it can be recommended for use. Potency data for all kinases with the structurally equivalent cysteine should be reported.

Sep 12 2016 - 6:55pm

This is an irreversible probe that inhibits JAK3 kinase activity. Although selectivity across the kinome was not assessed, the probe exhibits very good selective for JAK3 over JAK1, JAK2 and TYK2. The probe also exhibits good selectivity over kinases with a cysteine in a similar position.

Oct 26 2016 - 7:51am

Importantly, the in vivo effects of this probe on IL-2-driven proliferation were shown to be JAK3-dependent by using a mutant JAK3 (C905S JAK3), which exhibits similar activity to wild-type JAK3 but is not inhibited by their probe.

Oct 26 2016 - 7:57am

JAK3i is an irreversible probe molecule with good selectivity over closely related kinases. However, the paper lacks broader kinase selectivity data and this should be generated, especially for kinases with a cysteine in a structurally similar position, prior to use in cells.

Apr 7 2017 - 1:36pm

It would be helpful to see a more detailed selectivity assessment, but JAK3i appears reasonably selective with ~50-fold selectivity over BTK. The use of a C905S mutant to rescue demonstrates a clear JAK3-dependent effect for the compound. I would prefer further characterization of the compound to feel comfortable interpreting experimental results in any context that lacks a C905S mutant control. Additional PK/PD data would be valuable to guide use of the compound in vivo.

Apr 11 2017 - 11:52am

JAK3i

Probe Information

Target Details

JAK-3_HUMAN, JAKL, L-JAK, LJAK, JAK3, Janus kinase 3

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?

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JAK3i

Probe Information

Target Details

JAK-3_HUMAN, JAKL, L-JAK, LJAK, JAK3, Janus kinase 3

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?