Heterobifunctional PROTAC Degraders Targeting KRAS G12C via VHL E3 Ligase Recruitment: Degradation Kinetics, Selectivity Proteomics, and Anti-Tumor Efficacy in NSCLC Xenografts

This study investigates PROTAC-mediated degradation of oncogenic KRAS G12C via VHL-directed ubiquitination, characterizing degradation kinetics, selectivity by quantitative proteomics, and in vivo anti-tumor efficacy in NSCLC xenograft models within the context of chemical biology and oncology drug discovery, an area of growing scientific importance given its implications for KRAS G12C NSCLC treatment beyond sotorasib resistance, PROTAC linker optimization framework, and targeted protein degradation selectivity proteomics methodology. Using PROTAC synthesis (8 linker variants, 3-8 PEG units), KRAS protein degradation by western blot and quantitative TMT proteomics, DC50 and Dmax kinetic characterization, VHL-dependent rescue experiments, and SW1573 NSCLC xenograft efficacy with tumor volume measurement 28 days, we examine PROTAC bridging KRAS G12C (via covalent GDP-state lock) and VHL E3 ligase in a ternary complex that ubiquitinates KRAS for proteasomal degradation, achieving catalytic degradation below KRAS IC50 and overcoming resistance to direct inhibition by eliminating protein rather than blocking function in 8 PROTAC linker variants in H358 (KRAS G12C), SW1573 (KRAS G12C), and HCT116 (KRAS WT control) cell lines (n=3 biological replicates per condition); in vivo SW1573 xenograft: 10 mice per arm (vehicle, AMG510, optimal PROTAC P-5) at 10 mg/kg twice-weekly IP drawn from Pacific Oncology Institute BSL-2 cell culture facility, Thermo TMT 11-plex quantitative proteomics on Orbitrap Fusion Lumos, and athymic nude mouse xenograft facility with digital caliper tumor measurement. Results indicate that optimal PROTAC P-5 (5-unit PEG linker) achieves DC50 48 nM, Dmax 92.4%, Dmax within 4h; quantitative proteomics shows >10-fold selectivity (only KRAS G12C degraded at DC50, no off-targets >1.5-fold); xenograft tumor volume 28% vs. vehicle control, vs. 52% for AMG510 (p < 0.001), with DC50 48 nM; Dmax 92.4%; tumor 28% vs. vehicle; vs. 52% AMG510 as the primary quantitative benchmark. Concordance between primary and confirmatory measurement approaches exceeded 93%, validating the analytical framework. These findings contribute empirically to chemical biology and oncology drug discovery and carry actionable implications for the design of programs and policies targeting KRAS G12C NSCLC treatment beyond sotorasib resistance, PROTAC linker optimization framework, and targeted protein degradation selectivity proteomics methodology.