Fig 2: Cocrystal structure of GNE-2861/PAK4 and ATP/PAK4 that guided the structural optimization. (A) Focused regions in the structural optimization and SAR analyses. (B) Superimposition of GNE-2861 (wheat, PDB ID: 4O0V) and PF-3758309 (green, PDB ID: 2X4Z). (C) Superimposition of GNE-2861 (wheat, PDB ID: 4O0V) and CZh-226 (yellow, PDB ID: 5XVG). (D) Superimposition of GNE-2861 (wheat, PDB ID: 4O0V) and ATP (cyan, PDB ID: 4FIE). Related residues are shown as corresponding sticks. Red dashes represent hydrogen bonds.

Fig 3: Cocrystal structure of 41 and PAK4. (A) X-ray cocrystal structure of 41 bound to PAK4 (PDB ID: 7CMB, 2.3 Å resolution) and associated 2|Fo|−|Fc| omit map contoured at 1.0 σ level. (B) PAK4 is shown as a surface. Compound 41 is shown as a cyan stick structure. The key residues are shown as brown sticks. Red dashed lines indicate hydrogen bonds to key amino acids.

Fig 4: Cocrystal structure of 47/PAK4 and 55/PAK4. (A) X-ray cocrystal structure of 47 bound to PAK4 (PDB ID: 7CP3). Compound 47 is shown as a cyan stick structure. The key amino acids are shown as brown sticks. Red dashed lines indicate hydrogen bonds to key amino acids. (B) X-ray cocrystal structure of 55 bound to PAK4 (PDB ID: 7CP4). Compound 55 is shown as a green stick. The key amino acids are shown as yellow sticks. Red dashed lines indicate hydrogen bonds to key amino acids. (C) QM-torsion angle scans for 4-(6-ethynyl-1H-indol-1-yl)pyrimidin-2-amine (55a as a model molecule in QM-torsion angle scans) under M06-2X/6-311G∗∗. The baseline energy is obtained by subtracting the lowest energy of the compound.

Fig 5: Compound 55 suppresses the migratory and invasive potential of tumor cells. (A) An MTT assay showed the antiproliferative effect of compound 55 on A549, B16-F10 and B16-BL6 cells. (B) Compound 55 inhibits migration of A549, B16-F10 and B16-BL6 cells in wound healing assays. The migration relative ratio (C) was recorded. Transwell assay was performed to show the effect of indicated concentrations of compound 55 on A549, B16-F10 and B16-BL6 cells migration (D) and invasion (E), and migrated cells were counted, respectively (F, G). (H) Effects of compound 55 on total PAK4, p-PAK4(S474), LIMK1, p-LIMK1(T508) levels with 24 h treatment in A549 cells. The results are presented as the mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs control.