A bioorthogonal antifungal system driven by an all-in-one tetrazine reagent for precise imaging and therapy

Invasive fungal infections pose a serious threat to human health, highlighting the urgent need to develop novel antifungal therapy. Herein, we report the first bioorthogonal antifungal prodrug strategy that enables the activation of the Amphotericin B (AmB) prodrug and fluorescence visualization of fungal infections. We designed a fungal cell-targeting fluorescent tetrazine (Tz) agent, Tz-calcofluor white (CFW). This innovative Tz-CFW selectively binds to the fungal cell wall and aggregates within fungal cells for fluorescence visualization. Kinetic experiments demonstrated a rapid and efficient release via the inverse-electron-demand Diels–Alder reaction between the trans -cyclooctene (TCO)-caged AmB prodrug and Tz-CFW. The release of active AmB exhibited high specificity and potent in vitro activity against Candida albicans cells and Cryptococcus neoformans cells. In vivo studies demonstrated that TCO-AmB was selectively activated at the fungal infection site, reducing the fungal burden and inflammatory cytokine levels comparable to AmB. Thus, this pioneering bioorthogonal antifungal strategy could facilitate the development of precision medicine for antifungal therapy. We report the first bioorthogonal antifungal prodrug system, in which an all-in-one tetrazine agent, Tz-CFW, enables simultaneous active targeting of fungal cells, fluorescence visualization, and bioorthogonal activation of the TCO-AmB prodrug.