JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules

Area of Science:

• Biochemistry
• Structural Biology
• Chemical Biology

Background:

• The pseudokinase Janus kinase 2 (JAK2) JH2 domain is a key allosteric regulatory site.
• Developing selective inhibitors for JAK2 is crucial for treating myeloproliferative neoplasms and inflammatory diseases.
• Understanding the binding interactions at the JAK2 JH2 allosteric site is essential for drug discovery.

Purpose of the Study:

• To develop and validate a competitive fluorescence polarization (FP) assay for determining binding affinities at the JAK2 JH2 allosteric site.
• To report the synthesis of novel fluorescent probes (compounds 5 and 6) for use in the FP assay.
• To elucidate the binding poses of known JAK2 inhibitors within the JAK2 JH2 domain using X-ray crystallography.

Main Methods:

• Competitive fluorescence polarization (FP) assay development.
• Synthesis of fluorescent probes (compounds 5 and 6).
• Determination of dissociation constants (Kd) for ten compounds.
• X-ray crystallography of JAK2 JH2 in complex with selected inhibitors.

Main Results:

• A robust FP assay was established for quantifying ligand binding to the JAK2 JH2 allosteric site.
• Dissociation constants (Kd) were determined for ten compounds, including clinically relevant inhibitors.
• X-ray crystal structures provided detailed insights into the binding modes of NVP-BSK805, filgotinib, and compound 8 within the JAK2 JH2 domain.

Conclusions:

• The developed FP assay is a valuable tool for screening and characterizing small molecules targeting the JAK2 JH2 allosteric site.
• Structural data confirms the binding interactions of key inhibitors, aiding in the design of more potent and selective JAK2 modulators.
• This work contributes to the understanding of JAK2 allosteric regulation and supports the development of novel therapeutics.