The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor

Abstract

JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 Å crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family. IntroductionThe Janus kinases (JAKs) are an important family of intracellular protein tyrosine kinases (PTKs), with 4 mammalian members, JAK1, JAK2, JAK3, and TYK2, 1-5 as well as homologs in chicken, 6 fish, 7 and Drosophila. 8 The JAKs play critical roles in several important intracellular signaling pathways, including the eponymous JAK/STAT pathway, 9 central to the mediation of cytokine signaling. 10,11 It is this pivotal role in cytokine signaling that underpins the notion that specific JAK inhibitors may be therapeutically deployed in situations where cytokine activity results in disease. Important examples of this include autoimmune diseases such as rheumatoid arthritis and psoriasis, 12,13 myeloproliferative syndromes such as polycythemia vera, 14-17 leukemias, 18-20 lymphomas, 21 and cardiovascular disease 22,23 inter alia.Members of the JAK family each share a characteristic domain structure, 2 with a C-terminal PTK domain (known as the JAK homology-1 [JH1] domain), immediately adjacent to a kinase-like domain (JH2), and 5 additional JAK homology domains (JH3-JH7). While the JH2 domain appears to possess an important regulatory role on the PTK activity of the JH1 domain, 24-29 the precise mechanism by which this control is exerted is currently poorly understood. The role of a putative SH2-like domain (JH3/JH4) 2,30 is also unknown at present, whereas the function of a well-defined band F ezrin-radixin-moesin homology (FERM) domain (JH7) 31,32 appears to be critical for interaction of the JAKs with their cognate receptors and regulatory proteins.The JAKs coordinate specifically to different receptors, for example, JAK3 appears to be associated with cytokine receptors that include the ␥c chain of the interleukin-2 (IL-2) receptor (eg, IL-4, IL-7, etc), whereas JAK2 is associated with a wide range of cytokine receptors, including those activated by growth horm...

Glycogen synthase kinase-3β and p38 phosphorylate cyclin D2 on Thr280 to trigger its ubiquitin/proteasome-dependent degradation in hematopoietic cells

“…As shown in Figure 1a, IL-3 stimulation of IL-3-starved 32Dcl3 cells induced expression of cyclin D2 with concurrent phosphorylation of pRb, which was observed as early as 3 h after stimulation and increased gradually thereafter. Pretreatment with Jak inhibitor I (Jak-I) (Lucet et al, 2006), which inhibits the Jak2 kinase activated by IL-3, abrogated IL-3-induced cyclin D2 expression and pRb phosphorylation. Because Jak2 plays a crucial role in activation of various downstream signaling pathways, including the STAT5, mitogenactivated protein kinase kinase/extracellular signalregulated kinase (MEK/Erk) and PI3K/Akt pathways, from the IL-3 receptor (Ihle, 1995), we also examined the effects of inhibition of the PI3K/Akt and MEK/Erk pathways separately.…”

STAT3 Inhibition Overcomes Temozolomide Resistance in Glioblastoma by Downregulating MGMT Expression

“…However, STAT3 phosphorylation and MGMT expression did not completely synchronize compared with STAT3 inhibitor VI. This might be because JAK inhibitor I also inhibited Jak1, Jak2, Jak3 and Tyk2 and blocked STAT5 (51), and these off-target effects possibly inhibited the decrease of MGMT.…”

SB1518, a novel macrocyclic pyrimidine-based JAK2 inhibitor for the treatment of myeloid and lymphoid malignancies

“…The favorable preclinical data described here as well as the emerging clinical data with SB1518 and others such as TG101348, indicate that JAK1 activity is not mandatory for therapeutic benefit in MPDs. [18][19][20] It remains to be determined how the presence or absence of JAK1 activity contributes to a different clinical efficacy and safety profile. An additional feature of SB1518 is the potent activity against FLT3 and its drug-resistant D835Y mutant, but without activity against class III RTKs in the same concentration range.…”