NLRP3/CCR2 Blockade by Arglabin and Its Derivative Suppresses Myeloid-Driven Immunosuppression and Potentiates Anti-PD-L1 Immunotherapy in Hepatocellular Carcinoma

Background Hepatocellular carcinoma (HCC) evolves within an NLRP3-enriched immunosuppressive microenvironment where myeloid-derived suppressor cells (MDSCs) orchestrate T cell dysfunction. While the NLRP3 inhibitor arglabin (Arg) shows clinical anti-tumor activity, its immune-specific targets and mechanisms remain undefined. Method NLRP3 and CCR2 expressions were explored in tumor tissues from 14 HCC patients. Overall survival and the correlation between two proteins in HCC patients were analyzed using GEO and TCGA databases. Molecular interactions were validated through CETSA, SPR, and LC-MS/MS. In vivo antitumor efficacy was assessed in syngeneic murine models of H22 (BALB/c) and Hepa1-6 (C57BL/6) liver cancer cells. Functional analyses were conducted using CCK8, MDSC depletion (anti-Gr-1 antibody) and transwell invasion assays. Flow cytometry characterized TME composition, while in vivo bioluminescent imaging monitored tumor burden. Transcriptomic profiling of ChIP-qPCR deciphered CCR2 regulatory mechanisms. Structural optimization yielded compound C2, evaluated through pharmacokinetic studies and mechanism validation. Results NLRP3 overexpression in HCC patients correlated with reduced survival. Arg covalently bound to NLRP3 at Cys280 (KD = 16.32 nM), suppressing CCR2 transcription via non-canonical NLRP3 activity——independent of inflammasome activation. This myeloid-specific mechanism reduced MDSC infiltration (61.20% decrease vs control) while expanding cytotoxic CD8+ T cells (2.4-fold increase), achieving 56.42% tumor growth inhibition (TGI) in immunocompetent models. Synergy with anti-PD-L1 therapy enhanced TGI to 96.99%. Structural optimization generated orally C2, which maintained CCR2 suppression efficacy and MDSC suppression, and achieved 93.86% TGI at 18 mg/kg. Conclusion We redefine NLRP3 as a transcriptional regulator governing CCR2-dependent trafficking of MDSCs and identify Arg as a covalent NLRP3 inhibitor. The NLRP3/CCR2 axis emerges as a druggable target to alleviate immunosuppression in HCC, with Arg-derived C2 demonstrating significant clinical translation potential. This work establishes a framework for developing natural product-derived immunomodulators, which exhibit synergistic efficacy with immune checkpoint inhibitors to overcome resistance in cancer immunotherapy.