Generation of a chemical genetic model for JAK3

Abstract

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.

Reactivity-based chemical-genetic study of protein kinases

“…64 Additionally, in a recent work, Remenyi and colleagues described a chemical-genetic model in which a C905S mutant of JAK3 in mice was created to study the function of this kinase and its inhibitors in vivo . 82 A summary of the different cysteine residues that have been explored thus far in covalent chemical genetics with the respective protein kinases can be found in Fig. 9.…”

Docking and Selectivity Studies of Covalently Bound Janus Kinase 3 Inhibitors

“…Due to the highly conserved structural features of the ATP binding pocket, it has been challenging to achieve high selectivity among the JAK family. Many recent developments of JAK3 inhibitors have been focused on a JAK3 unique cysteine residue (CYS909) by forming a covalent bond with JAK3 inhibitors [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. The idea of developing an inhibitor that can covalently bind to cysteine 909 was from other covalent drugs such as afatinib, osimertinib, and ibrutinib ( Figure 2 ).…”

“…Three drugs in Figure 2 shared a common α,β-unsaturated amide moiety to allow the Michael addition to the target protein via covalent binding. A number of JAK3 covalently bound inhibitors have been reported and some have been studied under various stages of clinical trials [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ].…”