Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340 -356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.Syk is a tyrosine kinase that functions immediately downstream of antigen receptors in immune cells including B lymphocytes, mast cells, and macrophages (1-3). The central role of Syk in cell types associated with disorders such as rheumatoid arthritis and allergic rhinitis suggests that strategies to block Syk activation may have therapeutic benefit (4 -6). After receptor ligation and phosphorylation, Syk becomes localized to immune receptors and proceeds to phosphorylate downstream targets leading to Ca 2ϩ mobilization, initiation of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades, and activation of transcription factors such as NF-B (7). Although increased Syk activity is known to be indispensable for each of these cellular events, a molecular-level understanding of the steps leading to Syk activation has not been clearly defined.The Syk domain structure consists of an N-terminal pair of Src homology 2 (SH2) 2 domains separated by an inter-SH2 linker, an SH2-domain-kinase linker, and a C-terminal kinase domain. Recruitment of Syk to immune receptors involves binding of the tandem SH2 domains of Syk to motifs in the receptor known as immune tyrosine activation motifs (ITAMs), which are two YXXL sequences typically separated by 7-12 intervening residues (8, 9). Syk is known to have multiple ...
Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.
P38␣ is a protein kinase that regulates the expression of inflammatory cytokines, suggesting a role in the pathogenesis of diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus. Here, we describe the preclinical pharmacology of pamapimod, a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38␣ and p38 enzymatic activity, with IC 50 values of 0.014 Ϯ 0.002 and 0.48 Ϯ 0.04 M, respectively. There was no activity against p38␦ or p38␥ isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH 2 -terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC 50 , 0.06 M), but inhibition of JNK was not detected. Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) ␣ production by monocytes, interleukin (IL)-1 production in human whole blood, and spontaneous TNF␣ production by synovial explants from RA patients. LPS-and TNF␣-stimulated production of TNF␣ and IL-6 in rodents also was inhibited by pamapimod. In murine collagen-induced arthritis, pamapimod reduced clinical signs of inflammation and bone loss at 50 mg/kg or greater. In a rat model of hyperalgesia, pamapimod increased tolerance to pressure in a dose-dependent manner, suggesting an important role of p38 in pain associated with inflammation. Finally, an analog of pamapimod that has equivalent potency and selectivity inhibited renal disease in lupus-prone MRL/lpr mice. Our study demonstrates that pamapimod is a potent, selective inhibitor of p38␣ with the ability to inhibit the signs and symptoms of RA and other autoimmune diseases.
The development of a new series of p38α inhibitors resulted in the identification of two clinical candidates, one of which was advanced into a phase 2 clinical study for rheumatoid arthritis. The original lead, an lck inhibitor that also potently inhibited p38α, was a screening hit from our kinase inhibitor library. This manuscript describes the optimization of the lead to p38-selective examples with good pharmacokinetic properties.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.