Inhibitor Screen Identifies Covalent Inhibitors of the Protein Histidine Phosphatase PHPT1

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Protein histidine phosphatase PHPT1 plays a role in cellular signaling pathways.
• Limited chemical tools exist for studying histidine phosphorylation and dephosphorylation.
• Understanding PHPT1's biological roles is hindered by the lack of specific inhibitors.

Purpose of the Study:

• To identify the first chemical inhibitors of protein histidine phosphatase PHPT1 activity.
• To develop novel chemical probes for studying histidine phosphorylation.

Main Methods:

• A fluorogenic assay for PHPT1 activity was utilized.
• An inhibitor screen of approximately 4000 compounds was performed.
• Hit compounds were evaluated for selectivity against other phosphatases.

Main Results:

• Several selective PHPT1 inhibitors were identified from the compound screen.
• Norstictic acid emerged as the most potent inhibitor, with an IC50 of 7.9 ± 0.8 μM.
• Norstictic acid acts as a time-dependent, covalent inhibitor with KI = 90 ± 20 μM and kinact = 1.7 ± 0.1 min−1.

Conclusions:

• Norstictic acid is the first identified potent inhibitor of PHPT1.
• This compound provides a valuable tool for investigating PHPT1 function in biological systems.
• The findings open new avenues for exploring histidine phosphorylation in cellular processes.