Assay Development and Inhibitor Screening for Janus Kinase 2
Pölläniemi, Angelika (2023)
2023
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2023-05-02
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202304264506
https://urn.fi/URN:NBN:fi:tuni-202304264506
Tiivistelmä
Janus kinase 2 (JAK2) is an essential mediator of hematopoietic and growth signaling in cells. It signals as a part of the Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway, which ultimately regulates gene expression by mediating cytokine signaling from a diverse set of cytokines. Janus kinases are a family of four non-receptor tyrosine kinases that phosphorylate tyrosine residues located on their target proteins, such as STATs.
The aberrant function of JAK2 is implicated in blood-related malignancies, most notably myeloproliferative neoplasms (MPNs), where the loss of JAK2 autoinhibition leads to its constitutive activation, and subsequently, the excessive proliferation of myeloid cells.
Inhibitors of JAK2 are used as treatments for MPNs. These inhibitors target the ATP-binding site of the kinase, thus preventing kinase function by abrogating the binding of ATP. Current treatments have been shown to ease symptoms; however they do not have a curative effect. In addition, the inhibitors lead to the inadvertent inhibition of other kinases and eventually, resistance to treatment.
The development of allosteric JAK2 inhibitors could solve several issues with the current inhibitors. Allosteric inhibitors bind to less conserved areas on the protein as opposed to the conserved ATP-binding site. These allosteric binding sites have not been characterized due to the lack of identified allosteric JAK2 inhibitors.
Here, an assay measuring kinase activity is optimized to facilitate compound screening against the kinase domain of JAK2. The assay measures ADP that is produced by the kinase phosphorylation reaction. The extent of ADP production can be used to determine the level of kinase activity. The optimized assay is then used to screen a 5000-compound library against JAK2. Following the successful assay optimization and library screening, 13 hit compounds were identified that had an inhibitory effect on JAK2 activity. Further validation steps with alternative methods, such as fluorescence polarization and cell-based approaches, led to the identification of nine promising compounds, most of which bind JAK2 allosterically. As allosteric JAK2 inhibitors have not been described, identifying this many new and presumably allosteric compounds provides exciting prospects for further research, which may lead to the characterization of allosteric pockets and the development of novel treatments.
The aberrant function of JAK2 is implicated in blood-related malignancies, most notably myeloproliferative neoplasms (MPNs), where the loss of JAK2 autoinhibition leads to its constitutive activation, and subsequently, the excessive proliferation of myeloid cells.
Inhibitors of JAK2 are used as treatments for MPNs. These inhibitors target the ATP-binding site of the kinase, thus preventing kinase function by abrogating the binding of ATP. Current treatments have been shown to ease symptoms; however they do not have a curative effect. In addition, the inhibitors lead to the inadvertent inhibition of other kinases and eventually, resistance to treatment.
The development of allosteric JAK2 inhibitors could solve several issues with the current inhibitors. Allosteric inhibitors bind to less conserved areas on the protein as opposed to the conserved ATP-binding site. These allosteric binding sites have not been characterized due to the lack of identified allosteric JAK2 inhibitors.
Here, an assay measuring kinase activity is optimized to facilitate compound screening against the kinase domain of JAK2. The assay measures ADP that is produced by the kinase phosphorylation reaction. The extent of ADP production can be used to determine the level of kinase activity. The optimized assay is then used to screen a 5000-compound library against JAK2. Following the successful assay optimization and library screening, 13 hit compounds were identified that had an inhibitory effect on JAK2 activity. Further validation steps with alternative methods, such as fluorescence polarization and cell-based approaches, led to the identification of nine promising compounds, most of which bind JAK2 allosterically. As allosteric JAK2 inhibitors have not been described, identifying this many new and presumably allosteric compounds provides exciting prospects for further research, which may lead to the characterization of allosteric pockets and the development of novel treatments.