Targeting the Mutant JAK2 V617F Tyrosine Kinase by Small Molecules that Bind to its Pseudokinase Domain [electronic resource].

9780438970588

Ann Arbor : ProQuest Dissertations & Theses, 2018.

1 online resource (137 p.)

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Janus kinases (JAKs) are a family of non-receptor tyrosine kinases that signal via the conserved JAK-STAT cell signaling pathway. The four JAK family members (JAK 1-3 and TYK2) each consist of seven Janus homology (JH) domains that fold into an N-terminal FERM domain (JH7-JH5), an SH2-like domain (JH4-JH3), as well as tandem C-terminal pseudokinase (JH2) and tyrosine kinase (JH1) domains. Previous studies have demonstrated that the JAK pseudokinase domain is autoinhibitory of the protein's kinase domain activity, likely by direct interaction with the JH1 domain in cis. Various mutations within the JAK JH2 domain release autoinhibition, resulting in JAK hyperactivation. Specifically, the emergence of the mutant JAK2 V617F protein as the cause of the majority of BCR-ABL-negative myeloproliferative neoplasms (MPN) has made JAK2 an attractive target for therapeutic approaches. Several JAK2 inhibitors are currently in clinical trials, including momelotinib (CYT387) and pacritinib (SB1518), while the JAK1/2 inhibitor ruxolitinib has been FDA-approved for the treatment of myelofibrosis and resistant polycythemia vera. These JAKtargeted small molecule inhibitors used to treat MPNs are designed to target the JAK tyrosine kinase domain in an ATP-competitive manner. However, since the V617F mutation is localized within the JAK2 pseudokinase domain, these drugs do not discriminate between the wild type (WT) and mutant JAK proteins. Similarly, targeting JH1 leads to unwanted side-effects, which could theoretically be prevented should the small molecule selectively inhibit the mutant V617F protein. The pseudokinase domains of JAK1, JAK2, and TYK2 adopt a kinase fold and bind ATP with dissociation constants in the µM range. Recent mutagenesis studies suggest that displacing ATP from JAK2 JH2 reduces the basal and ligand-induced signaling of the full-length JAK2 V617F mutant protein while leaving the JAK2 WT unaffected. These experiments suggest that small molecule displacement of ATP from JAK2 JH2 may selectively inhibit the action of the oncogenic mutant JAK2 V617F protein. If this mutational data could be recapitulated pharmacologically, this could provide an avenue for selective inhibition of JAK2 V617F, In this work, I present independent studies used to identify small molecules that bind the JAK2 JH2 domain. In the first study (Chapter 3), a high-throughput in vitro fluorescence polarization screen (using a fluorescent-ATP molecule) was performed at the Yale Center for Molecular Discovery against the JH2 domain using several kinase inhibitor libraries. The top hit from the screen bound JAK2 JH1 and JH2 domains with high affinity. X-ray crystal structure analysis of this compound bound to JH1 and JH2 show a similar binding mode to both domains. In collaboration with the Jorgensen Laboratory in the Male Chemistry Department, medicinal chemistry efforts are underway in order to make this small molecule selective for JH2. The second study was performed in collaboration with the Jorgensen Laboratory from the Yale Chemistry Department. In this study (Chapter 4), the top hit from the fluorescence polarization screen mentioned above was used to develop a high affinity fluorescence polarization probe. Briefly, a fluorescein moiety was attached to the top hit and shown to bind to the JH2 domain with higher affinity than the previous fluorescent ATP probe. This has allowed for detection of binders to the JH2 domain with dissociation constants in the sub-µM range. The third study (Chapter 5) was performed in collaboration with Gilead Sciences. A high-throughput in silico screen was performed against the JH2 domain using internal Gilead kinase inhibitor libraries. Hits were identified and characterized using differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography. In the fourth study (Chapter 6), mutation of the JH2 gatekeeper residue caused decreased basal and ligand-induced activation of the JAK2 V617F mutant while leaving JAK2 WT unaffected. Mutation of a neighboring residue was able to recapitulate the mutational results of the gatekeeper mutation. These experiments reveal a potential allosteric mechanism underlying activation of the JAK2 V617F protein.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

August 21, 2019

Thesis (Ph.D.)--Yale University, 2018.

Yale University.