GSK583

Reagent Authentication Certificate:

Chemical Probes Portal GSK583.pdf

Protein target name:

RIPK2

Mechanism of action:

ATP competitive inhibitor

Recommended Concentration for use in cells:

200 nM

Probe Availability

MedChem Express (1346547-00-9)

Cayman (1346547-00-9)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

TKL

HUGO ID:

HGNC:10020

Entrez gene ID:

8767

UNIPROT ID:

O43353

Target alias:

Receptor-interacting serine-threonine kinase 2, CARD3, CARDIAK, RICK, RIP2

Probe Details

Chemical Probes Portal ID:

CPP1272

Probe alias:

GSK583; GTPL9134; SCHEMBL2595766; GSK'583; CS-5543; HY-100339

PubChem CID:

67469084

SMILES string:

CC(C)(C)S(=O)(=O)C1=CC2=C(C=CN=C2C=C1)NC3=NNC4=C3C=C(C=C4)F

InChi Key:

XLOGLWKOHPIJLV-UHFFFAOYSA-N

Physico-chemical properties

NMR:

1H NMR (400 MHz, DMSO-d6) δ ppm 13.00 (s, 1H), 9.96 (s, 1H), 9.19 (d, J = 1.5 Hz, 1H), 8.64 (d, J = 5.3 Hz, 1H), 8.07−8.11 (m, 1H), 7.98−8.04 (m, 1H), 7.60 (dd, J = 9.1, 4.0 Hz, 1H), 7.54 (dd, J = 9.2, 2.4 Hz, 1H), 7.33 (td, J = 9.0, 2.4 Hz, 1H), 7.28 (d, J = 5.6 Hz, 1H), 1.34 (s, 9H).

13C NMR (151 MHz, DMSO-d6) δ 155.7, 153.8, 150.5, 148.7, 141.4, 138.1, 130.6, 130.1, 128.5, 127.1, 118.3, 116.4, 116.0, 112.3, 104.2, 103.9, 59.3, 23.2.

Storage Recommendations

Storage (dry powder):

Ambient temperature, indefinite

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

5 nM

Potency assay:

A fluorescent polarization based binding assay was developed to quantitate interaction of novel test compounds at the ATP binding pocket of RIPK2 by competition with a fluorescently labeled ATP competitive ligand.

PDB ID for probe-target interaction (3D structure):

5J7B

Structure-activity relationship:

See J. Med. Chem. 2016, 59, 4867−4880.

Off target activity

Potency (off target):

IC50

Off target protein and potency:

RIPK3 16 nM

Potency assay (off target):

RIPK3: A fluorescent polarization based binding assay was developed to quantitate interaction of novel test compounds at the ATP binding pocket of RIPK3 by competition with a fluorescently labeled ATP competitive ligand.

Probe Selectivity in Vitro:

hERG: Electrophysiology; Whole cell voltage clamp technique permits investigation of the physiological role and pharmacological modulation of ion channels, transporters and receptors that link to ion channels. IC50 hERG=7.45 uM. P450 CYP3A4: Fluorescence/FLINT - Antagonism, pIC50 - Vivid Green Substrate Using Multidrop Combi Liquid Dispense, IC50 P450 CYP3A4=5 uM.

In cell validation

On target activity in cells

Potency in cells:

IC50

237 nM (human whole blood assay), 133 nM (rat whole blood assay), 18 nM (HEK cells)

Potency assay (cells):

Human and rat whole blood assays: MDP (muramyl dipeptide, NOD2 specific activating ligand) stimulated TNFalpha release measured in whole blood. HEK cells: MDP stimulated IL-8 release measured in HEK cells overexpressing NOD2.

Target engagement assay (cells):

Indirect

Off target activity in cells

Potency assay, off target (cells):

Cellular selectivity of GSK583 was evaluated in primary human monocytes. In the presence of 1 μM GSK583, NOD1- or NOD2-mediated cytokine production was completely inhibited (RIPK2-dependent pathways). In contrast, only weak inhibition was observed upon stimulation with ligands that selectively activate various TLR or cytokine receptors (RIPK2-independent pathways). The targeted receptors were stimulated with the following ligands: NOD1 (300 μg/mL, ieDAP), NOD2 (1 μg/mL, MDP), TLR2 (10 ng/mL Pam2Csk4), TLR4 (10 ng/mL, ultrapure LPS), TLR7 (10 μg/mL, gardiquimod), IL-1R (10 ng/mL, IL-1β), and TNFR (100 ng/mL, TNFα). Release of pro-inflammatory cytokines, either TNFα (NOD2, TLR2, TLR4, IL1R) or IL-8 (NOD1, TLR7, TNFR) weas measured by immunoassay.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

Cytoxicity was assessed in HepG2 cells: IC50=100 uM

In vivo validation

Organism:

Sprague-Dawley ratC57BL/6 mouse

Dose:

2 mg/kg oral, 1 mg/kg IV crossover (mouse and rat)

Route of delivery:

OralIntravenous

Plasma half life:

3.1 h (rat)2.1 h (mouse)

Systemic clearance:

15 mL/min/kg (rat), 13 mL/min/kg (mouse)

Fraction of probe bound to plasma protein:

97.4% bound in rat97.6% bound in human

Cmax:

260 ng/mL (rat)340 ng/mL (mouse)

Tmax:

1.3 h (rat)0.7 h (mouse)

Primary Reference

Reference (doi):

10.1021/acs.jmedchem.6b00211

Reference (PMID):

27109867

GSK583

Probe Availability

Probe Information

Target Details

Receptor-interacting serine-threonine kinase 2, CARD3, CARDIAK, RICK, RIP2

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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GSK583

Probe Availability

Probe Information

Target Details

Receptor-interacting serine-threonine kinase 2, CARD3, CARDIAK, RICK, RIP2

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?