Target identification of a macrocyclic hexaoxazole G-quadruplex ligand using post-target-binding visualization

Area of Science:

• Medicinal Chemistry
• Chemical Biology
• Molecular Biology

Background:

• Macrocyclic hexaoxazoles (6OTDs) are a class of G-quadruplex (G4) ligands.
• Certain derivatives, like L2H2-6OTD (1a), demonstrate cytotoxicity against cancer cells.
• Identifying the specific cellular targets of these ligands is crucial for understanding their mechanism of action.

Purpose of the Study:

• To identify the cellular target of the G4 ligand L2H2-6OTD (1a).
• To develop and apply a post-target-binding visualization strategy in living cells.

Main Methods:

• Utilized a click reaction (Huisgen cyclization) between an azide-conjugated ligand (L2H2-6OTD-Az, 1b) and a cell-permeable BODIPY-based dye (CO-1) with a strained alkyne.
• Employed Cu-free conditions for the click reaction.
• Confirmed the integrity of the azide-modified ligand and the unbiased localization of the dye.

Main Results:

• The azide modification did not alter the physical or biological properties of L2H2-6OTD (1a), including its anti-cancer activity.
• The post-binding visualization strategy successfully identified the cellular target.
• L2H2-6OTD (1a) was shown to colocalize with RNA G-quadruplexes in living cells.

Conclusions:

• The study successfully identified RNA G-quadruplexes as the cellular target of L2H2-6OTD (1a).
• The developed post-target-binding strategy is effective for visualizing ligand-target interactions in living cells.
• This finding provides insight into the anti-cancer mechanism of macrocyclic hexaoxazoles.