PF3758309

Reagent Authentication Certificate:

Chemical Probes Portal PF3758309.pdf

Protein target name:

PAK1, PAK4, PAK5, PAK6

Mechanism of action:

ATP-competitive inhibitor

Recommended Concentration for use in cells:

0.1 - 300 nM

Probe Availability

MedChem Express (898044-15-0)

Tocris (PF 3758309 dihydrochloride)

Cayman (898044-15-0)

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

STE

HUGO ID:

HGNC:8590 HGNC:16059 HGNC:15916 HGNC:16061

Entrez gene ID:

5058 10298 57144 56924

UNIPROT ID:

Q13153 O96013 Q9P286 Q9NQU5

Probe Details

Chemical Probes Portal ID:

CPP76

Probe alias:

PF-3758309

PubChem CID:

25227462

ChEMBL ID:

CHEMBL3128043

SMILES string:

CC1=NC2=C(C(=N1)NC3=NNC4=C3CN(C4(C)C)C(=O)N[C@H](CN(C)C)C5=CC=CC=C5)SC=C2

InChi Key:

AYCPARAPKDAOEN-LJQANCHMSA-N

Orthogonal probe:

GNE-2861

PAINs evaluation:

PAINs free

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

PAK4 18.7 nM

Potency assay:

KI: PAK5 18.1 nM, PAK6 17.1 nM, PAK1 13.7 nM PAK4 KD (ITC): 2.7 nM; (SPR) 4.5 nM

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

2X4Z

Off target activity

Potency assay (off target):

In kinase assays, PF-3758309 inhibited multiple off-target kinases of 146 tested: SRC, FYN, YES, AMPK, RSK, CHEK2, FLT3, PKC (multiple isoforms), PDK2, TRKA, AKT3, PRK1, FGR.

In cell validation

On target activity in cells

Potency in cells:

IC50

1.3 nM

Potency assay (cells):

Inhibition of PAK protein substrate phosphorylation

Target engagement assay (cells):

Indirect: inhibition of protein substrate phosphorylation

In vivo validation

Organism:

Athymic nude mice

Dose:

7.5-30 mg/kg

Route of delivery:

Oral

Organ of Interest

Organ (O):

xenograft

Target engagement assay:

Indirect: inhibition of protein substrate phosphorylation in xenograft tissue

Primary Reference

Reference (doi):

10.1073/pnas.0911863107

Reference (PMID):

20439741

SAB Rating

Rating for use in Cells

4 review(s)

Rating for use in Model Organisms

4 review(s)

SAB Members

SAB Comments

This probe meets minimal criteria for use in cellular and in vivo assays. It has additional biochemical activity in the PAK subfamily: PAK2: IC50 = 190 nM, PAK3: IC50 = 99 nM (PMID 20439741). No antiproliferative activity was noted in primary human hepatocytes: IC50 >1,000 nM (PMID 20439741). PF-3758309 was also used to inhibit PAK1 in tumor cells (PMID 23535073). It is classified as a poorly cell permeable compound using the MDCKII-LE assay and is a substrate of the efflux transporters P-gp and BCRP. Of note, a strong correlation was observed between P-gp (but not BCRP) expression in tumor cell lines and resistance to PF-3758309 in cellular assays. A similar, but weaker, correlation was observed in vivo (PMID 23524533).

Jul 3 2016 - 3:49pm

PF3758309 is reportedly potent against PAK4 (3-18 nM, depending on the assay format). Using a peptide phosphorylation assay, PF3758309 does not appear to be selective for PAK4 over PAK1, 5, and 6; it is 5-10x selective for PAK4 over PAK2-3.

Selectivity over the rest of the protein kinome remains a concern. PF3758309 was active (1 nM <IC50 <60 nM) against 13 of 146 kinases screened in a broad panel, including 3 SRC family kinases at 45-60 nM. The authors indirectly showed that SRC-family activity is not relevant for this compound when dosed in cells at 1 uM; however we do not have data about the remaining 10 liability kinases with IC50 <35 nM at 1 uM in cells. Furthermore, with only ~25% of the protein kinome screened, it is possible that ~40 kinases are inhibited with potencies IC50 <40 nM.

PF3758309 inhibits phosphorylation of a natural PAK4 substrate in cells with an IC50 ~1 nM and inhibits anchorage-independent growth (expected biology for PAK4 inhibition) at 240 pM. It is possible that PAK4 adopts a conformation in cells that binds PF3758309 more potently than PAK4 that is overexpressed and purified; however, it is very rare for an ATP competitive kinase inhibitor to be more potent in cells (competing with 2 mM ATP) than it is in in vitro biochemical (0.04 mM ATP) or a biophysical (0 mM ATP) assays. This is a signficant disconnect that is not resolved in the publication and may very well result from polypharmacology.

Use this compound with caution.

There is a reported allosteric PAK4 inhibitor that, while less potent, could be considered as a potentially more selective alternative (KPT-9274).

Sep 9 2016 - 10:26pm

PF-3758309 is reported as a potent, ATP-competitive inhibitor of PAK4 with additional activity against PAK1, PAK5 and PAK6, and, to a slightly lesser degree PAK3 and PAK2 (range of affinities PAK4 IC50=3 nM to PAK2 IC50=190 nM). PF-3758309 also shows binding to a number of representative kinases (20 with Ki <200nM) where there may be an overlapping relevant pharmacology. PF-3758309 clearly shows activity in both cellular and in vivo models at reasonable doses and using convenient routes of administration (oral & subcutaneous). PF-3758309 is commercially available, and there is a growing body of evidence with regard to its pharmacology and potential utility. If PF-3758309 is used, there needs to be a good understanding of both the on-target (PAKs) and off-target (other kinase activities) protein expression and function to interpret the results and draw a credible association between the compound, the target(s) and the outcomes.

Oct 19 2016 - 2:26pm

I would not recommend PF3758309 as a specific probe. While earlier data already have uncovered significant off-target activities, including potent CDK7 inhibition (J Med Chem 58, 111–129 (2015)), a newer study demonstrated that this compound inhibited the growth of Rosa26 and PAK4-KO melanoma cells equipotently (GI50 value of ~9 nM). Given that this drug is fully capable of killing cells in which its putative target has been deleted, the ability of PF-3758309 to block cancer cell growth must be through an off-target effect. (Lin, A. et al. Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Sci Transl Med 11, eaaw8412 (2019)).

Apr 1 2021 - 4:02pm

PF3758309

Probe Availability

Probe Information

Target Details

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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PF3758309

Probe Availability

Probe Information

Target Details

Probe Details

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?