IPA-3 is a selective non-ATP competitive Pak1 inhibitor with IC50 of 2.5 μM, no inhibition to group II PAKs (PAKs 4-6).

IPA-3 is a non-ATP-competitive, allosteric inhibitor of p21-activated kinase 1 (Pak1), with PIR3.5 serving as its control compound. It effectively blocks Cdc42-stimulated and sphingosine-dependent autophosphorylation of Pak1 on Thr423, without targeting the protein's exposed cysteine residues. The efficacy of IPA-3 is dependent on its disulfide bond; reduction by dithiothreitol (DTT) nullifies its inhibitory action on Pak1. Notably, IPA-3 obstructs Pak1 activation by various activators but does not affect already preactivated Pak1; it also impedes PDGF-induced Pak activation in mouse embryonic fibroblasts. This inhibition is partially achieved through covalent binding to Pak1's regulatory domain, an interaction that is both time- and temperature-sensitive, and prevents Cdc42 engagement. Additionally, IPA-3 demonstrates a direct bond to the Pak1 autoregulatory domain and offers reversible inhibition of PMA-induced membrane ruffling in cells.

Pak1 (150 nM final) is pre-incubated with MBP (8.3 μM), indicated proteins, and IPA-3 or DMSO in Kinase buffer for 20 minutes at 4°C. Cdc42-GTPγS (3.2 μM) is then added and the reaction is pre-equilibrated 10 minutes at 30°C. Kinase reactions are started by the addition of ATP (to 30 μM) containing [32P]ATP and are incubated 10 min and analyzed by SDS-PAGE and autoradiography.

Human primary schwannoma cells are grown on 96 well plates for 2 days. Cells are left untreated or treated with 5 μM IPA-3, 20 μM IPA-3 or 20 μM PIR-3.5 for 24 hours. The MTS-solution is left on the cells for 3 hours, before the absorbance at 490 nm is measured. The experiments are conducted three times and mean and standard error of the mean is calculated with Excel.