Janus kinases (JAKs) regulate hematopoiesis via the cytokine-mediated JAK-STAT signaling pathway. JAKs contain tandem C-terminal pseudokinase (JH2) and tyrosine kinase (JH1) domains. The JAK2 pseudokinase domain adopts a protein kinase fold and, despite its pseudokinase designation, binds ATP with micromolar affinity. Recent evidence shows that displacing ATP from the JAK2 JH2 domain alters the hyperactivation state of the oncogenic JAK2 V617F protein while sparing the wild type JAK2 protein. In this study, small molecule binders of JAK2 JH2 were identified via an screen. Top hits were characterized using biophysical and structural approaches. Development of pseudokinase-selective compounds may offer novel pharmacological opportunities for treating cancers driven by JAK2 V617F and other oncogenic JAK mutants.

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent and used in the assay are reported as well as results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine elucidate the binding poses.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

With standard scoring methods, top-ranked compounds from virtual screening by docking often turn out to be inactive. For this reason, metadynamics, a method used to sample rare events, was studied to further evaluate docking poses with the aim of reducing false positives. Specifically, virtual screening was performed with Glide SP to seek potential molecules to bind to the ATP site in the pseudokinase domain of JAK2 kinase, and promising compounds were selected from the top-ranked 1000 based on visualization. Rescoring with Glide XP, GOLD, and MM/ GBSA was unable to differentiate well between active and inactive compounds. Metadynamics was then used to gauge the relative binding affinity from the required time or the potential of mean force needed to dissociate the ligand from the bound complex. With consideration of previously known binders of varying affinities, metadynamics was able to differentiate between the most active compounds and inactive or weakly active ones, and it could identify correctly most of the selected virtual screening compounds as false positives. Thus, metadynamics has the potential to be a viable postprocessing method for virtual screening, minimizing the expense of buying or synthesizing inactive compounds. ■ TARGET PROTEIN JAK2 mutants are of interest for drug development because of JAK's role in the JAK−STAT pathway. 19 JH2, the pseudokinase Special Issue: New Trends in Virtual Screening

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