Development of an enzyme-coupled activity assay for Janus kinase 2 inhibitor screening

Area of Science:

• Biochemistry
• Pharmacology
• Molecular Biology

Background:

• Janus kinase 2 (JAK2) is crucial for signaling pathways involving cytokines like growth hormone and erythropoietin.
• The discovery of the JAK2 V617F mutation in 2005 highlighted its role in myeloproliferative neoplasms (MPNs).
• Current JAK2 inhibitors improve MPN patient quality of life but do not achieve molecular remission, necessitating novel therapeutic strategies.

Purpose of the Study:

• To develop a versatile, fluorescence-based activity assay for screening JAK2 inhibitors.
• To screen a library of natural products for novel JAK2-targeting compounds.
• To characterize the binding modes and selectivity profiles of identified inhibitors.

Main Methods:

• Development and implementation of a fluorescence-based JAK2 activity assay.
• Screening of a diverse set of small molecule natural products using the developed assay.
• Comparative analysis of assay performance against differential scanning fluorimetry.
• Profiling of identified hits against other Janus kinase (JAK) family members.

Main Results:

• Identification of 37 potential JAK2 inhibitor 'hits' from the natural product library.
• Most potent hits exhibited non-ATP competitive binding modes.
• Identified compounds demonstrated distinct selectivity profiles across JAK family members.
• The fluorescence assay showed consistency, simplicity, and cost-effectiveness.

Conclusions:

• The developed fluorescence assay is a reliable tool for screening diverse compound classes against JAK family members.
• Novel natural product-derived compounds targeting JAK2 were identified, offering potential for new MPN therapies.
• The assay facilitates the discovery of inhibitors with varied binding mechanisms and selectivity profiles.