Taxonomic and mechanistic insights into gut microbiota bioaccumulation of entacapone using bioorthogonal drug labelling.

Aim: The gut microbiota plays a key role in shaping individual responses to drugs, but current tools have limited potential to probe drug-microbe interactions within the complex, individualised gut environment. This study employed bioorthogonal labelling to track and identify gut microbial taxa and molecular mechanisms involved in the bioaccumulation of entacapone, a Parkinson's disease drug. Methods: We synthesised alkyne-tagged derivatives of entacapone and evaluated their suitability as molecular probes in ex vivo incubations with faecal communities or different Escherichia coli (E. coli) strains. Following incubation, tagged drugs were conjugated to a fluorescently labelled azide via click chemistry. Labelled cells were visualised, quantified, sorted via fluorescence-activated cell sorting (FACS), and identified via 16S ribosomal RNA (rRNA) gene amplicon sequencing. Results: Entacapone alkyne derivatives retained the biological activity and effects of the original drug on the microbiota, significantly reducing microbial loads and shifting community composition across the three donors tested. Conjugation of alkyne-entacapone with a labelled azide revealed that between 80% to 96% of all microbial cells in a donor's faecal sample accumulate entacapone. Nearly all taxa detected in incubations were recovered in labelled FACS fractions, confirming widespread uptake of the drug. Finally, we demonstrate that different E. coli strains exhibit varying levels of entacapone accumulation and identify a siderophore transporter that plays a role in this process. Conclusion: Our findings reveal that entacapone is widely bioaccumulated by the gut microbiota across three donors and identify a key molecular mediator of this accumulation. This study expands the toolkit for investigating drug-microbiome interactions and holds significant potential to advance our understanding of drug-microbiome dynamics and therapeutic outcomes.