# Advanced RPL19-TRAPKI-seq method reveals mechanism of action of bioactive compounds

## TL;DR

A new method called RPL19-TRAPKI-seq helps uncover how bioactive compounds work by analyzing translating mRNA in cells.

## Contribution

The RPL19-TRAPKI-seq method combines CRISPR/Cas9 and TRAP to accurately detect translating mRNA and reveal drug mechanisms.

## Key findings

- RPL19-TRAPKI-seq successfully validated rapamycin's mechanism of action via mTOR inhibition.
- SBF-1 was found to kill tumor cells by disrupting oxidative phosphorylation.
- The method provides a precise representation of translating mRNA, reflecting protein expression levels.

## Abstract

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 enrichment. 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 rapamycin, 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 investigated 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.

The online version contains supplementary material available at 10.1007/s13659-025-00500-3.