Abstract: CDC42 GTPase is overexpressed in multiple tumors and drives oncogenic progression by activating signaling pathways associated with cell proliferation, angiogenesis, and metastasis. To address the limitations of current methods for detecting cell division cycle 42 （CDC42）activation status, this study developed a high-throughput screening system based on bioluminescence resonance energy transfer （BRET）technology for rapid assessment of CDC42 activation. By coupling CDC42 with renilla luciferase （RLUC）and PAK1 with enhanced green fluorescent protein （EGFP）, a BRET detection system was constructed. Systematic optimization of protein fusion strategies （N-terminal or C-terminal fusion of RLUC/EGFP）and dual-plasmid transfection ratios （nine combinations）revealed that the combination of CDC42-C-EGFP with PAK1-C-RLUC （9∶1 ratio）significantly enhanced BRET signals, increasing the BRET ratio from 0.05 to 0.25 （4-fold increase）, thereby substantially improving detection sensitivity. Using this system, concentration-dependent inhibitory effects of known inhibitors （ML141, ARN25062, ARN24928）were validated. Among these, ARN25062 exhibited the strongest inhibitory potency and also demonstrated the strongest suppression of hepatocellular carcinoma cell proliferation, migration, and invasion, consistent with cell-based assay results. These findings confirmed that the established BRET system effectively characterized CDC42 inhibitor activity. The optimized BRET-based platform provided a critical tool for investigating CDC42 regulatory mechanisms and developing targeted therapeutics, while offering novel strategies for functional studies and drug discovery of small GTPases （e.g., Rho family proteins）.

Key words: CDC42, small G protein, BRET, ARN25062, ML141