A photoreactive small-molecule probe for 2-oxoglutarate oxygenases

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• 2-oxoglutarate (2-OG)-dependent oxygenases play critical roles in human physiology.
• Dysregulation of 2-OG oxygenases is implicated in diseases such as cancer, anemia, and ischemic conditions.
• Targeting 2-OG oxygenases presents a therapeutic strategy for various human diseases.

Purpose of the Study:

• To develop and validate a novel small-molecule probe for studying 2-oxoglutarate (2-OG)-dependent oxygenases.
• To demonstrate the probe's utility in identifying and profiling 2-OG oxygenase inhibitors.
• To explore the potential of small-molecule probes in substrate identification for oxygenases.

Main Methods:

• Design and synthesis of a small-molecule probe incorporating a hydroxyquinoline template, photoactivable crosslinking group, and affinity-purification tag.
• In vitro studies with recombinant 2-OG oxygenases.
• Application of the probe to human crude cell extracts and nuclear extracts (HEK293T).
• Demonstration of crosslinking to endogenous 2-OG oxygenases, including FBXL11 (KDM2A).

Main Results:

• The developed small-molecule probe successfully crosslinked to 2-oxoglutarate (2-OG)-dependent oxygenases in recombinant proteins.
• The probe demonstrated crosslinking capabilities with 2-OG oxygenases present at endogenous levels in human crude cell extracts.
• Specific crosslinking to the histone demethylase FBXL11 (KDM2A) was observed in HEK293T nuclear extracts, validating its use in inhibitor profiling.

Conclusions:

• The novel small-molecule probe is effective for studying 2-oxoglutarate (2-OG)-dependent oxygenases in complex biological samples.
• This probe serves as a valuable tool for inhibitor profiling of 2-OG oxygenases.
• The approach shows promise for future substrate identification studies of these enzymes.