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A PAK1 inhibitor
G-5555 is an inhibitor of p21-activated kinase 1 (PAK1; Ki = 3.7 nM), a non-receptor serine/threonine kinase involved in tumorigenesis.1 It is selective for PAK1 over the majority of targets in a panel of 235 kinases but does inhibit PAK2, PAK3, KHS1, LCK, MST3, MST4, SIK2, and YSK1 by greater than 70% with IC50 values ranging from 9 to 52 nM. G-5555 inhibits phosphorylation of MEK, a downstream target of PAK1, in EBC1 cells (IC50 = 69 nM). G-5555 (10, 20, and 30 mg/kg) dose-dependently reduces phosphorylation of MEK in tumors from an H292 non-small cell lung cancer (NSCLC) mouse xenograft model. G-5555 inhibits hERG channels less than 50% at a concentration of 10 μM, indicating the potential for a low risk of QT interval prolongation and potentially fatal arrhythmias.2
1.Ndubaku, C.O., Crawford, J.J., Drobnick, J., et al.Design of selective PAK1 inhibitor G-5555: Improving properties by employing an unorthodox low-pKa polar moietyACS Med. Chem. Lett.6(12)1241-1246(2015) 2.Braga, R.C., Alves, V.M., Silva, M.F., et al.Tuning HERG out: Antitarget QSAR models for drug developmentCurr. Top. Med. Chem.14(11)1399-1415(2014)
Kinase experiment: | The 10 ?L assay mixtures contain 50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, 2 ?M FRET peptide substrate, and PAK enzyme (20 pM PAK1; 50 pM PAK2; 90 pM PAK4). Incubations are carried out at 22°C in black polypropylene 384-well plates. Prior to the assay, enzyme, FRET peptide substrate and serially diluted test compounds (G-5555) are preincubated together in assay buffer (7.5 ?L) for 10 minutes, and the assay is initiated by the addition of 2.5 ?L assay buffer containing 4x ATP (160 ?M PAK1; 480 ?M PAK2; 16 ?M PAK4). Following the 60-minute incubation, the assay mixtures are quenched by the addition of development reagent, and 1 hour later the emissions of Coumarin (445 nm) and Fluorescein (520 nm) are determined after excitation at 400 nm using an Envision plate reader[1]. |
Animal experiment: | Mice: 3 mice in each of the two groups are administered 25 mg/kg oral suspension dose twice, with the second dose given 6 hours after the first dose. The dose volumes are 5 mL/kg for the IV group and 10 mL/kg for the PO groups. Following administration of G-5555 (12), 15 ?L of blood is collected at each time point are stored at -70 to -80°C until analysis[1]. |
参考文献: [1]. Ndubaku CO, et al. Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety. ACS Med Chem Lett. 2015 Oct 31;6(12):1241-6. |
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