Conversion of a False Virtual Screen Hit into Selective JAK2 JH2 Domain Binders Using Convergent Design Strategies.

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.