Small-molecules that covalently react with a human prolyl hydroxylase - towards activity modulation and substrate...

Area of Science:

• Biochemistry and enzymology
• Chemical biology
• Drug discovery

Background:

• Human prolyl hydroxylase domain 2 (PHD2) is a key regulator of cellular oxygen sensing and erythropoiesis.
• Dysregulation of PHD2 activity is implicated in various diseases, including anemia and cancer.
• Targeting PHD2 offers a therapeutic strategy for modulating hypoxia-inducible factor (HIF) signaling.

Purpose of the Study:

• To develop novel covalently binding modulators of human PHD2 activity.
• To establish a strategy for photocapture of PHD2 substrates using these modulators.
• To investigate the mechanism of covalent modification and its impact on enzyme function.

Main Methods:

• Synthesis and characterization of electrophile-bearing compounds designed for covalent interaction with PHD2.
• Biochemical assays to assess the inhibitory and covalent binding activity of the modulators.
• Site-directed mutagenesis to identify the specific residue involved in covalent modification.
• Photocapture experiments to validate the strategy for substrate identification.

Main Results:

• Novel modulators were identified that exhibit reversible active site binding to PHD2.
• These modulators undergo subsequent covalent reaction with lysine 408 (K408) in the flexible C-terminal region of PHD2.
• The covalent modification of PHD2 at K408 did not abolish its catalytic activity.
• The developed strategy showed promise for the photocapture and identification of PHD2 substrates.

Conclusions:

• Covalently binding modulators targeting PHD2 can be designed to retain enzyme activity.
• Modification of K408 represents a novel mechanism for PHD2 modulation.
• The photocapture strategy offers a powerful tool for studying PHD2 interactions and identifying its substrates.
• These findings provide a foundation for developing targeted therapeutics and research tools for PHD2-related pathways.