Molecular basis of JAK kinase regulation guiding therapeutic approaches: Evaluating the JAK3 pseudokinase domain as a drug target.

Janus kinases (JAK1-3, TYK2) are critical mediators of cytokine signaling and their role in hematological and inflammatory and autoimmune diseases has sparked widespread interest in their therapeutic targeting. JAKs have unique tandem kinase structure consisting of an active tyrosine kinase domain adjacent to a pseudokinase domain that is a hotspot for pathogenic mutations. The development of JAK inhibitors has focused on the active kinase domain and the developed drugs have demonstrated good clinical efficacy but due to off-target inhibition cause also side-effects and carry a black box warning limiting their use.

JAK inhibitor selectivity: new opportunities, better drugs?

Cytokines function as communication tools of the immune system, serving critical functions in many biological responses and shaping the immune response. When cytokine production or their biological activity goes awry, the homeostatic balance of the immune response is altered, leading to the development of several pathologies such as autoimmune and inflammatory disorders. Cytokines bind to specific receptors on cells, triggering the activation of intracellular enzymes known as Janus kinases (JAKs).

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface.

Development of an enzyme-coupled activity assay for Janus kinase 2 inhibitor screening.

JAK2 transmits signals of several important cytokines, such as growth hormone and erythropoietin. The interest toward the therapeutic targeting of JAK2 was boosted in 2005, when the somatic JAK2 V617F mutation, responsible for the majority of myeloproliferative neoplasms (MPNs) was discovered. JAK2 inhibitors have been approved for MPN therapy and they are effective in alleviating symptoms and improving the quality of life of the patients, but they do not lead to molecular remission.

Identification of Novel Small Molecule Ligands for JAK2 Pseudokinase Domain.

Hyperactive mutation V617F in the JAK2 regulatory pseudokinase domain (JH2) is prevalent in patients with myeloproliferative neoplasms. Here, we identified novel small molecules that target JH2 of JAK2 V617F and characterized binding via biochemical and structural approaches. Screening of 107,600 small molecules resulted in identification of 55 binders to the ATP-binding pocket of recombinant JAK2 JH2 V617F protein at a low hit rate of 0.

Tofacitinib treatment modulates the levels of several inflammation-related plasma proteins in rheumatoid arthritis and baseline levels of soluble biomarkers associate with the treatment response.

The data on the effects of tofacitinib on soluble proteins in patients with rheumatoid arthritis (RA) is currently very limited. We analyzed how tofacitinib treatment and thus inhibition of the Janus kinase-signal transducer and activation of transcription pathway affects the in vivo levels of inflammation-related plasma proteins in RA patients. In this study, 16 patients with active RA [28-joint disease activity score (DAS28) >3.

Tofacitinib Suppresses Several JAK-STAT Pathways in Rheumatoid Arthritis and Baseline Signaling Profile Associates With Treatment Response.

Objective: Current knowledge on the actions of tofacitinib on cytokine signaling pathways in rheumatoid arthritis (RA) is based on studies. Our study is the first to examine the effects of tofacitinib treatment on Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathways in patients with RA.

Methods: Sixteen patients with active RA, despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), received tofacitinib 5 mg twice daily for three months.

Mutation Is Associated with Increased STAT1 and STAT5 Activation in Two Family Members with Inflammatory Conditions and Lymphopenia.

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional coregulator that has an important role in the regulation of the immune response. IRF2BP2 has been associated with the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway, but its exact role remains elusive. Here, we identified a novel clinical variant, c.

Drammatic breaks: Break recovery experiences as mediators between job demands and affect in the afternoon and evening.

The present study focused on within-workday recovery, which has received less scholarly attention than has recovery outside work. We examined six break recovery experiences (detachment, relaxation, autonomy, mastery, meaning and affiliation) as possible mediators between daily emotional job demands, positive and negative affect both in the afternoon and in the evening. We conducted a one-work week diary study (N = 107) among Finnish schoolteachers with three daily measurements per workday.

Relationships between recovery experiences and well-being among younger and older teachers.

Purpose: The study had three aims. We investigated, first, how six recovery experiences (i.e.

Hyperactivation of Oncogenic JAK3 Mutants Depend on ATP Binding to the Pseudokinase Domain.

Janus kinase 3 (JAK3) tyrosine kinase has a central role in the control of lymphopoiesis, and mutations in JAK3 can lead to either severe combined immunodeficiency or leukemia and lymphomas. JAK3 associates with the common gamma chain (γc) receptor and functions in a heteromeric signaling pair with JAK1. In IL-2 signaling JAK1 is the effector kinase for STAT5 phosphorylation but the precise molecular regulatory mechanisms of JAK1 and JAK3 and their individual domains are not known.

Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations.

Background: Janus kinases (JAKs; JAK1 to JAK3 and tyrosine kinase 2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia, and the mutations strongly concentrate in the regulatory pseudokinase domain Janus kinase homology (JH) 2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation and pathway selectivity.

The regulation of JAKs in cytokine signaling and its breakdown in disease.

The JAK-STAT signal transduction pathway is responsible for mediating signals of over fifty cytokines, growth factors and hormones. Signaling through the JAK-STAT pathway is regulated on multiple levels, including intramolecular regulation by the JAK pseudokinase domain, and intermolecular regulation by a host of regulatory proteins. The advent of accessible genomic tools have provided a wealth of information on disease-associated mutations in the JAK-STAT pathway and its regulatory components.

Nucleotide-binding mechanisms in pseudokinases.

Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades.