Simon W. Lee

A small molecule ubiquitination inhibitor blocks NF-κB-dependent cytokine expression in cells and rats

A small molecule inhibitor of NF-κB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) α-induced IκBα degradation in MM6 cells but not the degradation of β-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFα, interleukin-1β, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC50 values below 1 μm. Ro106-9920 also blocked TNFα production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require βTRCP but associates with IκBα and will ubiquitinate IκBα S32E,S36E (IκBαee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IκBαee ubiquitination with an IC50 value of 2.3 ± 0.09 μm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCFβTRCP, the putative E3 for IκBα ubiquitination. Ro106-9920 was observed to be selective for IκBαee ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFα-induced IκBα degradation and NF-κB activation.

Cutting Edge: IL-1 Receptor-Associated Kinase 4 Structures Reveal Novel Features and Multiple Conformations

IL-1R-associated kinase (IRAK)4 plays a central role in innate and adaptive immunity, and is a crucial component in IL-1/TLR signaling. We have determined the crystal structures of the apo and ligand-bound forms of human IRAK4 kinase domain. These structures reveal several features that provide opportunities for the design of selective IRAK4 inhibitors. The N-terminal lobe of the IRAK4 kinase domain is structurally distinctive due to a loop insertion after an extended N-terminal helix. The gatekeeper residue is a tyrosine, a unique feature of the IRAK family. The IRAK4 structures also provide insights into the regulation of its activity. In the apo structure, two conformations coexist, differing in the relative orientation of the two kinase lobes and the position of helix C. In the presence of an ATP analog only one conformation is observed, indicating that this is the active conformation.

The kinase activities of interleukin-1 receptor associated kinase (IRAK)-1 and 4 are redundant in the control of inflammatory cytokine expression in human cells

IRAK-1 and IRAK-4 are protein kinases that mediate signaling by Toll/IL1/Plant R (TIR) domain-containing receptors including the IL-1, IL-18, and Toll-like receptors (TLRs). Although well studied in mouse systems, the mechanism by which they function in human systems is less clear. To extend our knowledge of how these proteins regulate inflammatory signaling in human cells, we genetically and pharmacologically manipulated IRAK-1 and IRAK-4 kinase activities in vitro. Ablation of IRAK-4 expression in human umbilical vein endothelial cells (HUVEC) with siRNA suppressed IL-1beta induced IL-6 and IL-8 production whereas IRAK-1 siRNA suppressed TNFalpha induced but not IL-1beta induced cytokine production. Complementation of IRAK-4-depleted cells with a kinase-inactive allele restored IL-1beta induced cytokine gene expression suggesting that the IRAK-4 kinase activity is dispensable relative to its scaffolding function. Consistent with this finding, an IRAK-4 selective kinase inhibitor (RO6245) that inhibited IRAK-1 degradation failed to block IL-1beta induced cytokine production. In contrast, an inhibitor of both IRAK-1 and IRAK-4 (RO0884) reduced IL-1beta induced p38 MAP kinase, c-Jun N-terminal kinase activation, and IL-6 production in HUVEC. RO0884 also antagonized IL-1beta, TNFalpha, and TLR-mediated cytokine production in human fibroblast-like synoviocytes and peripheral blood mononuclear cells. Therefore in human cells the non-kinase functions of IRAK-4 are essential, whereas the kinase activity of IRAK-4 appears redundant with that of IRAK-1. Pharmacologic inhibition of both kinases appears necessary to block pro-inflammatory cytokine production.

Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures.

Brutons tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X‐ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly‐rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2‐kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 310‐ and α‐helices, one of which collapses into the adenosine‐5′‐triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG‐out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure‐based design of specific inhibitors.

IL‐6 Is the Principal Factor Produced by Synovia of Patients with Rheumatoid Arthritis That Induces B‐Lymphocytes to Secrete Immunoglobulins

First, IL-6 is produced by synovial tissue of patients with rheumatoid arthritis (RA) and is the principal mediator produced by that tissue inducing differentiation of B-lymphocytes into antibody-forming cells. The Leu-1+ subset of B-lymphocytes is induced by IL-6 to secrete rheumatoid factor (IgM with anti-Fc gamma specificity). Second, the main cell types producing IL-6 in cells dissociated from RA synovial tissue are mononuclear leukocytes. Connective tissue type cells (synoviocytes) cultured from RA synovial tissue produce IL-6 in response to IL-1 beta, and IL-6 formation is increased by TGF-beta. Glucocorticoids strongly inhibit and PGE2 slightly inhibits IL-1-induced IL-6 mRNA expression in synoviocytes. Production of IL-6 increases when undissociated RA synovial tissue is maintained in culture, thus suggesting release from inhibition by a factor or factors not yet identified. Third, the major known local effect of IL-6 in RA synovial tissue is the augmentation of antibody formation and the major known systemic effect is the induction of the synthesis by the liver of acute-phase proteins, especially C-reactive protein. Levels of circulating C-reactive protein are reported to decrease in RA patients receiving long-acting antirheumatic drugs, which would be consistent with the interpretation that immature monocyte-derived macrophages are major producers of IL-6 in these patients.

Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase

Brutons tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr551 within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr551 causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 ± 20 μm and KmS1 = 37 ± 8 μm. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a “substrate clamping” type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.

Highly potent, non-basic 5-HT6 ligands. Site mutagenesis evidence for a second binding mode at 5-HT6 for antagonism.

A series of 5-HT(6) ligands derived from (R)-1-(amino)methyl-6-(phenyl)sulfonyltetralin was prepared that yielded several non-basic analogs having sub-nanomolar affinity. Ligand structure-activity relationships, receptor point mutation studies, and molecular modeling of these novel ligands all combined to reveal a new alternative binding mode to 5-HT(6) for antagonism.

Activated human langerhans cells express mRNA for IL-1α and IL-1β and produce these cytokines but do not secrete them

Abstract Human Langerhans cells (LC) were isolated from epidermal cell preparations by panning with mouse anti-CD1 monoclonal antibody. RNA was prepared and probed for the presence of mRNAs for various cytokines using radiolabeled cDNAs. After stimulation with phorbol myristate acetate LC express RNA for interleukin 1α (IL-1α) and interleukin 1β (IL-1β) and produce proteins but do not secrete them at detectable levels. LC-associated IL-1, particularly IL-1α, may play a role in antigen presentation. PMA did not induce IL-6 expression in LC. The addition of lipopolysaccharide, a muramyl dipeptide analog, ionomycin, IL-1α, tumor necrosis factor-α, insulin-like growth factor-1 or IL-6 did not induce IL-1 mRNA in LC. UVB augmented IL-1β mRNA expression. Glucocorticoids did not detectably affect IL-1α or IL-1β mRNA levels following PMA induction, however, staurosporin inhibited IL-1β mRNA synthesis. Thus the inducers and regulators of IL-1 formation in human LC and monocytes are not identical.