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).

PAK1:2.5 μM

IPA-3是一种非ATP竞争性的、异构体抑制剂，针对p21激活的蛋白激酶1 (Pak1)。PIR3.5作为IPA-3的对照化合物。IPA-3阻止由Cdc42刺激的Pak1在Thr423上的自磷酸化。同时，IPA-3也阻断了鞘氨醇依赖的Pak1自磷酸化，并且不针对Pak1上的裸露半胱氨酸残基。IPA-3的二硫键对于抑制Pak1至关重要，还原剂二硫苏糖醇（DTT）通过体外还原作用消除了IPA-3对Pak1的抑制。IPA-3能抑制多种激活剂引发的Pak1活化，但不影响已经激活的Pak1。在小鼠胚胎成纤维细胞中，IPA-3抑制了由PDGF触发的Pak激活。[1] IPA-3部分通过与Pak1调节域的共价结合来抑制Pak1的活化。IPA-3在特定时间和温度下与Pak1形成共价结合，阻断Pak1激活剂Cdc42的结合，并直接与Pak1自调节域结合。IPA-3可逆地抑制PMA诱导的细胞膜皱褶形成。[2]

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.