Covalent Modification of the JH2 Domain of Janus Kinase 2

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 attach-ment 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. 30 -fold 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.

Automated summary

This study reports the development of probe molecules that can covalently modify the JAK2 pseudokinase domain (JH2). The researchers aimed to selectively target the JH2 domains over the kinase domains (JH1) as potential therapeutic targets. They found that the JH2 domains of JAK1, JAK2, and TYK2 have a cysteine residue in the catalytic loop that is not present in their JH1 domains. By attaching substituents terminating in acrylamide warheads to modify Cys675 of JAK2 JH2, the researchers successfully achieved covalent attachment. The binding selectivity of the JAK2 JH2 domain over the JH1 domain increased by approximately 30-fold after a 20-hour incubation.