Computer-aided drug design, synthesis and identification of disulfide compounds as novel and potential allosteric PAK1 inhibitors

Abstract

p21-activated kinase 1 (PAK1) is an evolutionarily conserved serine/threonine protein kinase, which has been considered as one of the key regulatory factors in signaling network of tumor cells. Therefore, inhibition of PAK1 may be a potential approach to treat many types of solid tumors. Several allosteric inhibitors of PAK1 have been identified, and the most well known one is IPA-3. But its biological activity is not satisfied, and the structure activity relationship (SAR) of PAK1 allosteric inhibitors is unclear. In this study, we designed and synthesized 13 potential allosteric inhibitors by using computer-aided drug design based on the structure of the existing PAK1 allosteric inhibitors. All the compounds were characterized by ¹H-NMR and ¹³C-NMR, among which six were not reported previously. SAR was investigated by pharmacological studies and In03 and In06 showed increased PAK1 inhibition than previously reported IPA-3. These findings could guide further structure optimization of PAK1 inhibitors.

Fig. 1. Field point template of IPA-3 and its analogs in Template 1. The negative fields (blue point) and the hydrophobic fields (orange point) were common pharmacophore features of these three compounds while the positive charge (red point) and the van der Waal's surface (yellow point) were not.

Fig. 2. The structure of IPA-3 and the compounds we designed.

Fig. 3. Field point patterns of the synthetic compounds. The common pharmacophore features of them were similar to that of IPA-3. (A) Aligning results of the compounds we designed. (B) Pharmacophore features of the compounds we designed.

Fig. 4. In03 and In06 have better vitro potency than IPA-3 to inhibit PAK1 kinase activity. Recombinant PAK1 was incubated with disulfide compounds followed by addition of Cdc42-GTP and MBP in the presence of ATP. Luminescence assay was performed 3 times in duplicate. #, P < 0.01.

Fig. 5. In03 and In06 also suppress the phosphorylation of PAK1 Thr212. BGC823 cells were incubated with disulfide compounds and western blot was performed with PAK1 Thr212 antibody to detect the activity of PAK1.

Fig. 6. In03 and In06 inhibit the activity of PAK1 kinase in BGC823 cell. BGC823 cells were incubated with disulfide compounds and the lysates were immunoprecipitated with PAK1 antibody. Kinase assay was performed to assess the ability of disulfide compounds in inhibiting PAK1 kinase.

Fig. 7. Ribbon diagram of the PAK1 protein structure (PDB code: 1F3M). Dash line represents the missing residues need to be constructed.

Fig. 8. Ramachandran plot for modified PAK1.

Fig. 9. Docking of In03, In06 and IPA-3 with PAK1 protein structure. The binding modes of In03 and In06 with PAK1 protein structure were similar to that of IPA-3. (A) The binding site of In03, In06 and IPA-3. (B) Docking interaction of In06 with PAK1. (C) Docking interaction of In03 with PAK1. (D) Docking interaction of IPA-3 with PAK1.