摘要Natural products play a crucial role in new drug development,but their druggability is often limited by uncertain molecular targets and insufficient research on mechanisms of action.In this study,we developed a new RPL19-TRAPKI-seq method,combining CRISPR/Cas9 and TRAP technologies,to investigate these mechanisms.We identified and validated seven ribosomal large subunit surface proteins suitable for TRAP,selecting RPL19 for its high enrich-ment.We successfully established a stable cell line expressing EGFP-RPL19 using CRISPR knock-in and verified its efficiency and specificity in enriching ribosomes and translating mRNA.Integrated with next-generation sequencing,this method allows precise detection of translating mRNA.We validated RPL19-TRAPKI-seq by investigating rapamy-cin,an mTOR inhibitor,yielding results consistent with previous reports.This optimized TRAP technology provides an accurate representation of translating mRNA,closely reflecting protein expression levels.Furthermore,we investi-gated SBF-1,a 23-oxa-analog of natural saponin OSW-1 with significant anti-tumor activity but an unclear mechanism.Using RPL19-TRAPKI-seq,we found that SBF-1 exerts its cytotoxic effects on tumor cells by disturbing cellular oxidative phosphorylation.In conclusion,our method has been proven to be a promising tool that can reveal the mechanisms of small molecules with greater accuracy,setting the stage for future exploration of small molecules and advancing the fields of pharmacology and therapeutic development.