GNE-2861

Protein target name:

PAK4, PAK5, PAK6

Mechanism of action:

Inhibitor

Recommended Concentration for use in cells:

10-50 uM (see author note in Comments)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

STE

HUGO ID:

HGNC:16059 HGNC:15916 HGNC:16061

Entrez gene ID:

10298 57144 56924

UNIPROT ID:

O96013 Q9P286 Q9NQU5

Target alias:

p21 (RAC1) activated kinase 4, p21 (RAC1) activated kinase 5, PAK7, p21 (RAC1) activated kinase 6,

Probe Details

Chemical Probes Portal ID:

CPP1572

Probe alias:

2OL

PubChem CID:

68181954

ChEMBL ID:

CHEMBL3128042

SMILES string:

OC1(C#CC2=CC=C3N=C(NCCOC)N(C4=NC(N)=NC=C4)C3=C2)CCCCC1

InChi Key:

RZXMIHOUHYSGJO-UHFFFAOYSA-N

Orthogonal probe:

PF-3758309

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

30 uM

Potential reactivities:

Unknown

NMR:

1H NMR (500 MHz, DMSO-d6) δ 8.43 (d, J = 5.5 Hz, 1H), 8.22 (t, J = 5.5 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.25 (m, 1H), 7.16 (m, 1H), 7.14 (s, 2H), 6.91 (d, J = 5.5 Hz, 1H), 5.36 (s, 1H), 3.60 (overlapping m, 4H), 3.28 (s, 3H), 1.88 1.19 (overlapping m, 10H); 13C NMR (126 MHz, DMSO) δ 163.5, 161.9, 157.0, 155.3, 144.0, 131.8, 127.3, 116.3, 113.9, 113.8, 100.3, 93.3, 84.2, 70.7, 67.4, 58.5, 42.5, 25.4, 23.3.

Storage Recommendations

Storage (dry powder):

Room temperature

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

PAK4: 3.3 nM

Potency assay:

Activity of human recombinant PAK4 (KD, kinase domain) protein assessed through measuring the phosphorylation of a FRET peptide substrate.

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

4O0T

Structure-activity relationship:

PAK4 SAR was determined by using the FRET phosphorylation assay. Data for 15 compound are shown (see J Med Chem paper), covering structural modifications at three different sites.

Off target activity

Potency (off target):

Off target protein and potency:

PAK1: 2.9 uM

Potency assay (off target):

We assessed percentage inhibition at 100 nM using a 222-membered kinase panel (Invitrogen). All except PAK4,5,6 were inhibited <50%. Comparable results were obtained for 6 kinases with a Met gatekeeper.

In cell validation

On target activity in cells

Potency in cells:

EC50

10 uM

Potency assay (cells):

Cell viability in MDA-MB-436 and MCF10A PIK3CA (H1047R) cells. GNE-2861 dose-dependently inhibited cell migration in wound healing assays with the same cells (Essen Bioscience Incucyte platform).

Toxicity

Cytoxicity assay:

Primary Reference

Reference (doi):

10.1021/jm401768t

Reference (PMID):

24432870

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

no rating

SAB Members

SAB Comments

Author's note: GNE-2861 is a potent and exquisitely kinase-selective Group II PAK inhibitor, and to the best of my knowledge, it is the most selective inhibitor for this target group reported in the literature. The biochemical PAK4 Ki is 3 nM, and no significant inhibition of other kinases was observed at a concentration of 100 nM in a panel of 222 kinases. A drawback is the lack of cellular target engagement data (phosphorylation of PAK4/5/6 substrates) for this probe. The high permeability as assessed in MDCK cells (MDCK Papp (A-B) = 22.2 x 10^-6 cm/s) suggests that this probe should be able to enter cells, but obviously there is no experimental proof in the context of relevant cancer cell lines. The available cell data are limited to cellular efficacy data (wound healing and proliferation), and high drug concentrations are needed to impart effects in these assays (at least 10 uM). While this raises potential concerns, it is also possible that there isn't an efficient translation from molecular target engagement to a functional response in the specific contexts investigated, and this probe might be more efficacious in other functional settings and in combination with other targeted agents. While this is surely speculative, I believe that this probe nevertheless is more useful for assessing PAK4 biology than others that are available (e.g., PF-3758309, a promiscuous kinase inhibitor with questions on its true drivers of cellular efficacy).

May 30 2017 - 1:08pm

This is the best available probe for group II PAKs (4,5,6). The disconnect between enzyme (PAK4 IC50 8 nM) and cellular effect (effects observed at 10 uM & 50 uM) is highlighted by the authors as surprising but no explanation is offered. There is no attempt made to confirm target engagement in the cell assay, so it is difficult to comment further. Kinase selectivity (in an Invitrogen panel tested at 0.1 uM) is impressive, although it would have been good to include NIK data since at 1 uM it showed marginal activity. Nevertheless, the compound represents the best available probe for group II PAKs.

Jul 5 2017 - 2:35pm

GNE-2861 is a PAK4/5/6 inhibitor with isoform selectivity over group I PAKs (PAK1/2/3). The binding mode of the inhibitor is well investigated and gives an explanation of the partial isoform selectivity. Kinome selectivity was determined with a medium-sized panel consisting of 222 kinases. The screened concentration of 100vnM is considerably low (PAK4 IC50=8 nM) and, therefore, the selectivity data should be handled with caution. Cellular penetration was measured, and the compound showed an effect on the cell migration of several cancer cell lines with upregulated PAK signalling. These effects were shown at relatively high concentrations of 10-50 uM and a cellular PAK inhibition is not finally proven. When cellular target engagement is shown (e.g., by means of a PAK downstream phosphorylation assay), this compound may serve as a useful tool for the investigation of group II PAK signaling in cells.

Jul 5 2017 - 3:00pm

This is a thoroughly characterized probe and the supporting information is of high quality. The disconnect between in vitro and cellular potency, while potentially explicable and consistent with PAK4 inhibition, suggests that comparison to genetic modulation of PAK4 be included in cellular experiments to support conclusions regarding mechanism of action. Recommendation of a negative control by the authors would also increase utility.

Nov 4 2020 - 11:12am

GNE-2861

Probe Information

Target Details

p21 (RAC1) activated kinase 4, p21 (RAC1) activated kinase 5, PAK7, p21 (RAC1) activated kinase 6,

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

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GNE-2861

Probe Information

Target Details

p21 (RAC1) activated kinase 4, p21 (RAC1) activated kinase 5, PAK7, p21 (RAC1) activated kinase 6,

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?