Fig 2: IODVA1 inhibits lamellipodia and circular dorsal ruffle (CDR) formation in MDA-MB-231 cells and decreases Rac activation.(A) IODVA1 inhibits EGF-induced lamellipodia formation in MDA-MB-231 cells. MDA-MB-231 cells were plated on fibronectin-coated coverslips, serum starved for 4 h, incubated with the indicated concentrations of IODVA1 for 1 h, then EGF (50 ng/mL) stimulated, fixed and stained with Phalloidin Alexa Fluor 594 (F-Actin, pseudocolored red) and DAPI (nuclei, pseudocolored blue). Representative images show lamellipodia formation and enrichment of actin staining at the leading edge (white closed arrowheads) at 0 and 0.3 µM concentrations and lack of lamellipodia with rounded cell morphology at 1 and 3 µM. Note equally distributed phalloidin staining with presence of stress fibers in the cell body of the 1 and 3 µM treated-cells (white open arrowheads) indicative of stationary cells. Scale bar = 10 µm. Results are representative of three independent experiments. (B) IODVA1 inhibits PDGF-induced CDR formation in 3T3 fibroblasts. NIH-3T3 cells were plated on fibronectin-coated coverslips, serum starved for 4 h, incubated with the indicated concentrations of IODVA1 for 1 h, then PDGF (50 ng/mL) stimulated, fixed and stained with Phalloidin Alexa Fluor 594 (F-Actin, pseudocolored magenta) and DAPI (nuclei, pseudocolored blue). Closed white arrowheads indicate circular dorsal ruffles, arrows indicate lack of elongated morphology typical in stimulated fibroblasts. The percentage of cells with CDRs was counted as the number of cells with CDRs normalized to the total number of cells in the field. Around 150 cells were counted per condition per experiment. Cells with multiple CDRs were counted only once. Results are representative of three independent experiments. Scale bar = 10 µm. (C) MDA-MB-231 cells were incubated with IODVA1 (IO1) at the indicated concentrations for 1 h, lysed, and incubated with GST-PAK-GBD (binds active Rac and Cdc42) and GST-Rhotekin RBD (binds active RhoA). The protein complexes were resolved on SDS-PAGE and immunoblotted with pan-Rac, Cdc42 or RhoA antibodies. Levels of active Rac (RacGTP, % of control), active Cdc42 (Cdc42GTP, % of control) and active RhoA (RhoAGTP, % of control) were quantified using ImageJ and ImageLab and show combined data as mean ± s.e.m. from at least 3 independent experiments. (D) Left panel—MDA-MB-231 cells were incubated with IODVA1 (IO1, 1 µM) for 30 min, lysed, and immunoblotted for pPAK1(T423)/ pPAK2(T402). Lysates were loaded in two replicates. Results shown are mean ± s.e.m. of two independent experiments. Right panel–MDA-MB-231 cells were incubated with IODVA1 (IO1, 0.3 and 1 µM) for 30 min or 3 hours, lysed and immunoblotted for pPAK4(S474)/ pPAK5(S602)/ PAK6(S560). Results shown are mean ± s.e.m. of two independent experiments. n.s.–not significant, *—p < 0.05, **—p < 0.01, ****—p < 0.0001.

Fig 3: Schematic illustration of JNK-VEGFR2-RhoJ-PAK2/PAK4-BRAF-ERK pathway regulating GBM angiogenesis. JNK inhibitor SP600125 inhibits RhoJ expression level, the phosphorylation level of VEGFR2, and its downstream PAK and ERK1/2 signaling pathways, which regulate endothelial cell function and further impact GBM angiogenesis and progression.

Fig 4: The structures of representative PAK4 inhibitors.

Fig 5: 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.