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 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. 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.