Structure-Based Engineering of Irreversible Inhibitors against Histone Lysine Demethylase KDM5A

Area of Science:

• Biochemistry
• Enzymology
• Medicinal Chemistry

Background:

• Human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases share highly conserved active sites, complicating selective inhibitor development.
• A noncatalytic cysteine (Cys481) was identified near the active site of KDM5 histone H3 lysine 4 demethylases, a feature absent in other demethylase families.

Purpose of the Study:

• To explore the potential of Cys481 for selective inhibitor design against KDM5A.
• To synthesize and characterize novel thienopyridine-based inhibitors targeting Cys481 for covalent modification.

Main Methods:

• Synthesis of thienopyridine analogs (N70 and N71) designed for covalent interaction with Cys481.
• Characterization of inhibitory and binding activities of N70 and N71 against KDM5A.
• Determination of cocrystal structures of KDM5A catalytic domain with N70 and N71.

Main Results:

• Noncovalent inhibitor N70 showed αKG-competitive inhibition, reversible after dialysis.
• Covalent inhibitor N71 demonstrated enzyme concentration-dependent inhibition, persisting after dialysis, indicating covalent modification of Cys481.
• Cocrystal structures confirmed the binding modes of N70 and N71.

Conclusions:

• The identified Cys481 in KDM5A provides a viable target for developing selective covalent inhibitors.
• The synthesized N71 analog effectively targets and covalently modifies KDM5A, offering a promising strategy for selective enzyme inhibition.