Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins

Area of Science:

• Biochemistry
• Marine Biology
• Pharmacology

Background:

• Microcystins are potent cyanobacterial toxins with potential anticancer applications.
• Understanding their physiological roles and optimizing them for drug development requires advanced derivatization techniques.
• Previous attempts to modify microcystins via precursor-directed biosynthesis were unsuccessful.

Purpose of the Study:

• To develop "clickable" microcystin analogues for bioorthogonal reactions.
• To investigate the substrate acceptance of microcystin-producing cyanobacteria for unnatural amino acids.
• To assess the impact of modifications on microcystin cytotoxicity and cellular uptake.

Main Methods:

• Precursor-directed biosynthesis using azide- or terminal alkyne-containing amino acid analogues.
• Cultivation of microcystin-producing cyanobacteria strains with modified media.
• Conjugation of a clickable microcystin derivative with a fluorogenic dye.
• Cytotoxicity assays against cell lines expressing human organic anion transporting polypeptides (OATPs).
• Time-lapse microscopy to observe cellular uptake.

Main Results:

• Successfully generated over 40 different clickable microcystin analogues by varying unnatural amino acid incorporation.
• Demonstrated that neither the unnatural amino acid nor the fluorescent label significantly altered cytotoxicity against OATP1B1/1B3 expressing cells.
• Observed rapid cellular uptake of the fluorescent microcystin in cells expressing OATPs.

Conclusions:

• Precursor-directed biosynthesis is a viable method for creating functionalized microcystins.
• Clickable microcystins retain biological activity and can be used for cellular imaging and mechanistic studies.
• The findings support the potential of microcystins as anticancer drug leads and provide tools for their further investigation.