Abstract:

<p>The V617F mutation in the pseudokinase (JH2) domain of JAK2 is a frequent cause of myeloproliferative neoplasms (MPNs) and JAK2 inhibitors are an important therapeutic option for patients with these conditions. Currently approved JAK2 inhibitors target the kinase domain of JAK2, and while they exhibit varying degrees of selectivity among the four members of the JAK kinase family and across the kinome, all are equipotent against wild-type and V617F-mutant JAK2. Inhibition of WT JAK2 and other family members limits tolerability and therefore efficacy of current agents, making development of a mutant-selective JAK2 inhibitor a long-sought goal. Because the pseudokinase domain regulates the activity of the kinase domain and is the site of the V617F mutation, it represents a potential target for development of mutant-selective JAK2 inhibitors. Here we describe compounds that covalently bind the ATP-site of the JAK2 pseudokinase domain by targeting either Cys675 or Lys677, residues that are unique to the pseudokinase domain. In purified enzyme assays, we find that selected compounds potently inhibit pseudokinase-containing constructs with relative sparing of the isolated kinase domain, indicative of an allosteric mechanism of inhibition. Compound 20 (JH-XVII-135-2) incorporates a lysine-reactive fluorosulfate warhead and inhibits full-length JAK2 with an IC of 160&nbsp;nM. A co-crystal structure of 20 with the JAK2 pseudokinase reveals its binding mode and confirms covalent modification of Lys677, which was also observed using LC-MS/MS. Though not mutant-selective in our biochemical assays, 20 demonstrates proof-of-concept for allosteric inhibition of JAK2 via the pseudokinase domain and for covalent targeting of JAK2 on Lys677.</p>