Selective Janus Kinase 2 (JAK2) Pseudokinase Ligands with a Diaminotriazole Core

Abstract

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.

Metadynamics as a Postprocessing Method for Virtual Screening with Application to the Pseudokinase Domain of JAK2

“…The 30 compounds selected from the virtual screening, shown in Figure , encompassed a range of cores and interactions with the hinge and several other residues in the binding site as well as Glide SP scores from −11.8 to −9.9. Such low values seemed auspicious, since a selection of six known binders found previously through a high-throughput screen and seven more discovered in our laboratory (Figures and ) were prepared and docked; they yielded Glide SP scores of −10.6 to −6.5. For example, the pose for the highest-ranked compound from the SP scoring, JAK-198, is shown in Figure .…”

“…In contrast to the virtual screening and metadynamics simulations, wild-type JAK2 JH2 was used instead of the V617F variant because of its higher stability. As position 617 is remote from the binding site, significant differences in K d results are not observed for the mutant and the wild-type proteins. , Preparation and purification of the protein followed described procedures . JAK2 JH2 binding was measured by a competitive assay adapted from a previous study in our laboratory .…”

There’s more to death than life: Noncatalytic functions in kinase and pseudokinase signaling

“…The allosteric functions of pseudokinases have emerged as the predominant target for small-molecule interventions to date. Much interest has been shown in modulating the pseudokinase (JH2) domains of TYK2 and JAK2 to control their role as suppressors of the catalytic activity of the adjacent tyrosine kinase domain in each protein ( 97 , 98 , 99 ). Given its involvement in EGFR family signaling and cancer, HER3 has also emerged as a desirable pseudokinase target of therapeutic interest.…”

Janus Kinases in Leukemia

“…This is also indicated by mutagenesis experiments, where point mutations in the JH2 ATP-pocket and surrounding sites show selective inhibition of pathogenic signaling [28]. Several inhibitors have been designed against JAK2 JH2, some of which bind to the domain with high affinity [129][130][131]. However, these compounds have not shown any inhibition of JAK2 kinase activity.…”