GSK484

Reagent Authentication Certificate:

Chemical Probes Portal GSK484.pdf

Protein target name:

PADI4

Mechanism of action:

Mixed-mode inhibitor

Recommended Concentration for use in cells:

Up to 10 uM

Probe Availability

MilliporeSigma (1652591-81-5)

Cayman (1652591-81-5)

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Arginine deaminase

HUGO ID:

HGNC:18368

Entrez gene ID:

23569

UNIPROT ID:

Q9UM07

Target alias:

PAD, PAD4, PADI5, PDI4, PDI5

Probe Details

Chemical Probes Portal ID:

CPP149

Probe alias:

GTPL8577, GSK 484, GSK-484

PubChem CID:

86340175

SMILES string:

CN1C2=C(C=C(C=C2OC)C(=O)N3CC[C@H]([C@H](C3)N)O)N=C1C4=CC5=CC=CC=C5N4CC6CC6

InChi Key:

BDYDINKSILYBOL-WMZHIEFXSA-N

Control compound:

GSK106

PAINs evaluation:

PAINs free

Physico-chemical properties

Storage Recommendations

Storage (solution):

-20 oC 2 years

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

50 nM (no Ca2+), 250 nM (2 mM Ca2+)

Potency assay:

Fluorescence polarization binding assay

Off target activity

Potency (off target):

Ki, Kii

Off target protein and potency:

PADI1: Ki = 108 uM, Kii = 470 uMPADI2 Ki = 107 uM, Kii = 1350 uMPADI3 Ki = 2090 uM, Kii = 1230 uMPADI4 Ki = 6.8 uM, Kii = 65 uM

Probe Selectivity in Vitro:

Negligible activity in a panel of 50 protein kinases Negligible activity against COX2, MAOB, PDE4B, GPCRs, ion channels, transporters, AHR, NR1I2

In cell validation

On target activity in cells

Potency assay (cells):

Dose-dependent activity against target in human and mouse neutrophils detected in a citrullination assay

Target engagement assay (cells):

Indirect: citrullination assay

Primary Reference

Reference (doi):

10.1038/nchembio.1735

Reference (PMID):

25622091

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

GSK484 is a PADI4-selective in vitro chemical probe. PAD4 is a calcium-dependent enzyme which catalyzes the reaction in which protein arginine residues are converted into citrulline, with the release of ammonia. This pathway is anticipated to be relevant to multiple immune and inflammatory processes, oncology, and the regulation of stem cell maintenance. GSK484 reversibly binds to the low-calcium form of PAD4 (IC50 = 50 nM, mouse neutrophils) and has been shown to be selective over PAD1-3 in cellular assays and with assays employing recombinant enzymes. The probe has been studied in cells but has not been evaluated with in vivo models. It is not clear that the probe will function in vivo due to the need for low concentrations of calcium to render the target in optimal form for binding. This may be difficult to achieve in a physiological model.

Dec 26 2016 - 12:07pm

There is solid experimental evidence supporting GSK484 as a PAD4-specific inhibitor in biochemical systems, including nanomolar potencies in fluorescence polarization, ligand-competition, and NH3-release assays, X-ray crystallography, and convincing SAR. Users should note GSK484 is significantly more active in the absence of calcium-bound PAD4. Mass spectrometry and dialysis experiments support a reversible, competitive binding mechanism in the absence of calcium and a more complicated mode of action at higher calcium concentrations. 50-target biochemical activity profiling demonstrates reasonable selectivity compared to unrelated proteins in vitro. There is also solid experimental evidence supporting GSK484 as a relatively PAD4-specific inhibitor in certain cell-based systems, including Western blotting, cell imaging, and chemoproteomic mass spectrometry. Note there is an approximately 10-20-fold decrease in potency in most cell-based experiments, which could be due to a variety of factors including calcium status, intracellular concentration, and nonspecific protein binding, and parameters of the biochemical assays, among others. GSK484 is relatively nontoxic to certain cell lines at 10 uM. Users should also note that while the original manuscript presents biochemical and cellular selectivity data for PAD1-4 but not PAD6. There is an inactive analog, GSK106. I would highly recommend including this control compound in any experiments utilizing GSK484.

I recommend this probe for use in cell-based systems, with the caution to carefully consider calcium concentrations when designing and interpreting experiments. Depending on the experimental question, I would also consider monitoring the contributions of the related PDA1-3 and PDA6, since there is always potential for other PDA engagement in cell-based systems. The original report does not provide data regarding use in model organisms, so it is unclear how GSK484 may engage PDA4 in complex organisms given its calcium-dependent activity. I do not endorse this compound for use in animal models, though additional data demonstrating useful (e.g., potent, selective, consistent with proposed mechanism of action) target engagement in vivo may enable a future recommendation.

Jul 10 2017 - 12:05pm

GSK484

Probe Availability

Probe Information

Target Details

PAD, PAD4, PADI5, PDI4, PDI5

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Primary Reference

Supporting Organizations

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GSK484

Probe Availability

Probe Information

Target Details

PAD, PAD4, PADI5, PDI4, PDI5

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?