Phospho-GEF-H1 cellular assay

TR-293-KDG cells were constructed from HEK293 cells stably transfected with tetracycline-inducible PAK4-kinase domain (amino acids 291 ~ 591) and constitutively expressed HA-tagged GEFH1ΔDH (amino acids 210 ~ 921). TR-293-KDG cells were incubated for 3 hrs with PF-3758309, captured on an anti-HA antibody-coated plate, detected with an anti-phospho-S810-GEF-H1 antibody, and quantified with a horseradish peroxidase-goat anti-rabbit antibody conjugate.

Cell lines

HEK293T, HCT116 and SKOV3 cells

Preparation method

The solubility of this compound in DMSO is >24.5 mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

1 μM; 72 hrs

Applications

In tested cell lines, PF-3758309 inhibited phosphorylation of the PAK4 substrate GEF-H1 and anchorage-independent cell growth. In addition, PF-3758309 also inhibited accumulation of endogenous pGEF-H1 in HCT116 cells.

Animal models

A panel of human xenograft tumor models

Dosage form

7.5 ~ 30 mg/kg; p.o.; b.i.d., for 9 ~ 18 days

Applications

PF-3758309 significantly inhibited tumor growth in 5 models including HCT116 and A549 models which were PAK4-dependent. On the other hand, PF-3758309 showed no inhibition in DLD1 cells with a loss-of-function mutation in one of the PAK4 alleles.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

PF-3758309 is an inhbitor of PAK4 with IC50 of 1.3 nM [1].

P21-activated kinases (PAKs) are the family of serine/theronine kinases, which play important role in linking Rho GTPase to cytoskeleton reorganization and nuclear signaling. PAK4 is a member of PAKs family, specifically responsive for interacting with GTP bound form of Cdc42 and JNK family. PAK4 is involved in filopodia formation and may play a role in the reorganization of the actin cytoskeleton.

Biochemical study had identified that PF-3758309 was an ATP-competitive inhibitor of PAK4, which inhibited the kinase activity [1]. When PAK4 was screened with a panel of tumor cell lines, it was found PF-3758309 inhibited the phosphorylation of PAK4 substrate GEF-H1, and also the PAK4-induced anchorage-independent cell growth, with IC50 value of 1.3 nM and 4.7 ±3 nM respectively [1]. When PF-3758309 was screened with a panel of PAKs related kinases, it exhibited good potency and specificity for PAK4 [1]. Cell analysis confirmed that PF-3758309 modulates PAK4-dependent signaling nodes and identifies unexpected links to additional p53pathways [1].

In vivo activity of PF-3758309 was examined in a panel of human xenograft model, including HCT116 and A549 model. Twice daily oral administration of PF-3758309 (7.5-30 mg/kg BID) for 9-18 days resulted in significant inhibition of tumor growth in all the models. The tumor growth was shown to be caused by PAK-dependent pathways in HCT116 and A549 model. Additionally, PF-3758309 was shown to regulate PAK-associated cell proliferation and survival in certain models. Therefore, PF-3758309 exhibited inhibitory activity on PAK4 [1].

Reference:
[1] Murray B W et al. , Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth. Proc Natl Acad Sci USA. 2010, 107(20): 9446-9451.