JAK2 is a non-receptor tyrosine kinase that regulates hematopoiesis through the JAK-STAT pathway. The pseudokinase domain (JH2) is an important regulator of the activity of the kinase domain (JH1). V617F mutation in JH2 has been associated with the pathogenesis of various myeloproliferative neoplasms, but JAK2 JH2 has been poorly explored as a pharmacological target. In light of this, we aimed to develop JAK2 JH2 binders that could selectively target JH2 over JH1 and test their capacity to modulate JAK2 activity in cells. Toward this goal, we optimized a diaminotriazole lead compound into potent, selective, and cell-permeable JH2 binders leveraging computational design, synthesis, binding affinity measurements for the JH1, JH2 WT, and JH2 V617F domains, permeability measurements, crystallography, and cell assays. Optimized diaminotriazoles are capable of inhibiting STAT5 phosphorylation in both WT and V617F JAK2 in cells.
The Janus kinase 2 (JAK2) pseudokinase domain (JH2) is an ATP-binding domain that regulates the activity of the catalytic tyrosine kinase domain (JH1). Dysregulation of JAK2 JH1 signaling caused by the V617F mutation in JH2 is implicated in various myeloproliferative neoplasms. To explore if JAK2 activity can be modulated by a small molecule binding to the ATP site in JH2, we have developed several ligand series aimed at selectively targeting the JAK2 JH2 domain. We report here the evolution of a false virtual screen hit into a new JAK2 JH2 series. Optimization guided by computational modeling has yielded analogues with nanomolar affinity for the JAK2 JH2 domain and >100-fold selectivity for the JH2 domain over the JH1 domain. A crystal structure for one of the potent compounds bound to JAK2 JH2 clarifies the origins of the strong binding and selectivity. The compounds expand the platform for seeking molecules to regulate JAK2 signaling, including V617F JAK2 hyperactivation.
Probe molecules that covalently modify the JAK2 pseudokinase domain (JH2) are reported. Selective targeting of JH2 domains over the kinase (JH1) domains is a necessary feature for ligands intended to evaluate JH2 domains as therapeutic targets. The JH2 domains of three Janus kinases (JAK1, JAK2, and TYK2) possess a cysteine residue in the catalytic loop that does not occur in their JH1 domains. Starting from a non-selective kinase binding molecule, computer-aided design directed attachment of substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2. Successful covalent attachment was demonstrated first through observation of enhanced binding with increasing incubation time in fluorescence polarization experiments. Covalent binding also increased selectivity to as much as ca. 30fold for binding the JAK2 JH2 domain over the JH1 domain after a 20-h incubation. Covalency was confirmed through HPLC electrospray quadrupole time-of-flight HRMS experiments, which revealed the expected mass shifts.
The Janus kinases (JAKs) are key components of the JAK-STAT signaling pathway and are involved in myriad physiological processes. Though they are the molecular targets of many FDA-approved drugs, these drugs manifest adverse effects due in part to their inhibition of the requisite JAK kinase activity. However, the JAKs uniquely possess an integrated pseudokinase domain (JH2) that regulates the adjacent kinase domain (JH1). The therapeutic targeting of JH2 domains has been less thoroughly explored and may present an avenue to modulate the JAKs without the adverse effects associated with targeting the adjacent JH1 domain. The potential of this strategy was recently demonstrated with the FDA approval of the TYK2 JH2 ligand deucravacitinib for treating plaque psoriasis. In this light, the structure and targetability of the JAK pseudokinases are discussed, in conjunction with the state of development of ligands that bind to these domains. ■ SIGNIFICANCEThe JAK pseudokinase (JH2) domains present attractive targets for modulation of JAK-STAT pathways to circumvent adverse side effects of traditional Jakinibs. This has been demonstrated by the recent FDA approval of the TYK2 JH2 ligand deucravacitinib. Understanding the structure of these domains and the state of development of their ligands is important for future development of the JAK pseudokinases as therapeutic targets.
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