Identification of Potential Selective PAK4 Inhibitors Through Shape and Protein Conformation Ensemble Screening and Electrostatic-Surface-Matching Optimization

Abstract

P21-activated kinase 4 (PAK4) plays a crucial role in the proliferation and metastasis of various cancers. However, developing selective PAK4 inhibitors remains challenging due to the high homology within the PAK family. Therefore, developing highly selective PAK4 inhibitors is critical to overcoming the limitations of existing inhibitors. We analyzed the structural differences in the binding pockets of PAK1 and PAK4 by combining cross-docking and molecular dynamics simulations to identify key binding regions and unique structural features of PAK4. We then performed screening using shape and protein conformation ensembles, followed by a re-evaluation of the docking results with deep-learning-driven GNINA to identify the candidate molecule, STOCK7S-56165. Based on this, we applied a fragment-replacement strategy under electrostatic-surface-matching conditions to obtain Compd 26. This optimization significantly improved electrostatic interactions and reduced binding energy, highlighting its potential for selectivity. Our findings provide a novel approach for developing selective PAK4 inhibitors and lay the theoretical foundation for future anticancer drug design.

Keywords: MM/GBSA; P21-activated kinases; electrostatic complementarity; molecular docking; molecular dynamics; selective inhibitors.

Figure 1

Overall workflow.

Figure 2

Structural comparison of the binding cavities in PAK1 and PAK4 with their ligands. (a) Crystal structure of PAK4 (PDB ID: 7CP4) and (b) crystal structure of PAK1 (PDB ID: 5DEY). The hydrophobic surfaces of the binding cavities are visualized using color shading. The secondary structures of the receptors, including the α-helices, are highlighted: PAK1 is shown in blue (c) and PAK4 is shown in pink (d).

Figure 3

Chemical structures of representative inhibitors targeting PAK1 and PAK4.

Figure 4

Binding cavities of PAK1 and PAK4. Left: Crystal structure of the PAK4 (PDB ID: 7CP4). Right: Crystal structure of the PAK1 (PDB ID: 5DEY). The binding cavities of both receptors are highlighted using electrostatic potential coloring, with key binding site residues labeled in blue.

Figure 5

Heatmap of the free energy decomposition for PAK1 and PAK4 systems. Red indicates the interactions of inhibitors with key residues around the binding site in PAK1, while blue indicates the interactions of inhibitors with key residues around the binding site in PAK4. Key residues are highlighted in yellow boxes.

Figure 6

(a) The chemical structure of STOCK7S-56165, (b) the chemical structure of Compd 26, and (c) binding free energy contributions of Compd 55, STOCK7S-56165, and Compd 26 to PAK4 (energy unit: kcal/mol). The result for Compd 26 is the average value of stable 100-250 ns from three independent replicate simulations.

Figure 7

(a) RMSD analysis of Compd 26 during MD simulations from three independent replicate calculations, (b) 3D binding pose of Compd 26 with PAK4, where hydrogen bonds and hydrophobic interactions are represented by green and pink lines, respectively, and (c) IGMH analysis of the interaction between Compd 26 and PAK4; the green color block indicates that the main interaction is van der Waals interaction.

Figure 8

ESP surface of PAK4-binding site (PDB ID: 7CP4), Compd 55, STOCK7S-56165, and Compd 26.