Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance

Area of Science:

• Pharmacology
• Molecular Biology
• Drug Discovery

Background:

• Genetic resistance to kinase inhibitors limits durable therapeutic responses.
• Janus kinase 2 (JAK2) inhibitors are crucial for treating various conditions, but resistance is a significant challenge.

Purpose of the Study:

• To investigate a novel mechanism of JAK2 kinase inhibition by fedratinib that prevents the emergence of genetic resistance.
• To understand how fedratinib overcomes resistance mechanisms observed with other JAK2 inhibitors.

Main Methods:

• In vitro drug screening to identify resistance mutations.
• Structural modeling, mutagenesis, and biochemical studies to elucidate binding mechanisms.
• In vitro binding assays using purified proteins.

Main Results:

• Identified 211 amino acid substitutions conferring resistance to ruxolitinib and cross-resistance to other JAK2 inhibitors.
• These resistant variants remained fully sensitive to fedratinib.
• Fedratinib exhibits dual binding sites, with strong affinity for the substrate-binding site (Kd = 20 nM) and poor affinity for the ATP site (Kd = ~8 μM).
• Mutations in the substrate-binding pocket render the kinase catalytically incompetent, preventing resistance.

Conclusions:

• Fedratinib's dual-binding mechanism, particularly its interaction with the substrate-binding site, effectively prevents the emergence of genetic resistance.
• Targeting the substrate-binding site is a promising strategy for developing next-generation, resistance-free kinase inhibitors.