Insights on JAK2 Modulation by Potent, Selective, and Cell-Permeable Pseudokinase-Domain Ligands

Abstract

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.

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

“…Preliminary LCMS of the cell extracts indicates that the methyl ester of 11 is hydrolyzed to 11 , but that 11 is present in relatively minute quantities in the cell lysates as compared to the medium, indicating poor permeability. PAMPA does not appear to be a good predictor of cell permeability for this series, a phenomenon more clearly illustrated in our more developed triazole series . Permeability does appear to be a major contributing factor for poor cellular activity of the triazole series.…”

“…PAMPA does not appear to be a good predictor of cell permeability for this series, a phenomenon more clearly illustrated in our more developed triazole series. 35 Permeability does appear to be a major contributing factor for poor cellular activity of the triazole series. Nevertheless, the present work sets the stage for additional advances by providing molecules that bind strongly and selectively to JAK2 JH2 and a structural basis for their activity.…”

Covalent Modification of the JH2 Domain of Janus Kinase 2

“…The series of new analogues began by replacing the sulfonamide of 1 with an N-methylamide to improve cell permeability and reduce mass. 14 The resultant compound provided a baseline for the subsequent studies. Attachment of the acrylamide warhead was pursued at the 3-position of the difluorophenyl ring with methylene, ethylene, and ethyleneoxy linkers, and at the 4-position with an ethyl linker.…”

“…For TYK2 JH2, deucrava­citinib’s selectivity is achieved by a deuteromethyl­amide motif near the hinge region . In the non-covalent JAK2 JH2 ligands developed in our lab, − selectivity is promoted through a combination of a carboxylate that interacts with Thr555 and Arg715 and a terminal aryl pharmacophore that interacts with Arg715 and Trp718. , Acquiring selectivity is challenging for kinase inhibitors, owing to the abundance of kinases and the sequence conservation in their ATP-binding sites. Thus, the use of targeted covalent inhibitors (TCIs) has been receiving increased attention as an alternative approach to enable high kinase selectivity. − The Janus kinases are no exception, and covalent inhibitors acting on the JAK3 JH1 domain have been developed. − In addition, one compound, xanthatin, has been shown to bind covalently to Cys243 of the FERM domain of JAK2 …”

“…The series of new analogues began by replacing the sulfonamide of 1 with an N -methyl­amide to improve cell permeability and reduce mass . The resultant compound provided a baseline for the subsequent studies.…”