Jim W. Barnett

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.

Structural insights for design of potent spleen tyrosine kinase inhibitors from crystallographic analysis of three inhibitor complexes.

Spleen tyrosine kinase is considered an attractive drug target for the treatment of allergic and antibody mediated autoimmune diseases. We have determined the co‐crystal structures of spleen tyrosine kinase complexed with three known inhibitors: YM193306, a 7‐azaindole derivative and R406. The cis‐cyclohexyldiamino moiety of YM193306 is forming four hydrophobically shielded polar interactions with the spleen tyrosine kinase protein and is therefore crucial for the high potency of this inhibitor. Its primary amino group is inducing a conformational change of the spleen tyrosine kinase DFG Asp side chain. The crystal structure of the 7‐azaindole derivative bound to spleen tyrosine kinase is the first demonstration of a 2‐substituted 7‐azaindole bound to a protein kinase. Its indole‐amide substituent is tightly packed between the N‐ and C‐terminal kinase lobes. The co‐crystal structure of the spleen tyrosine kinase–R406 complex shows two main differences to the previously reported structure of spleen tyrosine kinase soaked with R406: (i) the side chain of the highly conserved Lys is disordered and not forming a hydrogen bond to R406 and (ii) the DFG Asp side chain is pointing away from and not towards R406. The novel protein–ligand interactions and protein conformational changes revealed in these structures guide the rational design and structure‐based optimization of second‐generation spleen tyrosine kinase inhibitors.

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.

Variations in aggrecan structure modulate its susceptibility to aggrecanases.

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

Crystal structures of IL-2-inducible T cell kinase complexed with inhibitors: insights into rational drug design and activity regulation.

IL‐2‐inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2‐mediated inflammatory diseases. By applying high‐throughput protein engineering and crystallization, we have determined the X‐ray crystal structures of IL‐2‐inducible T cell kinase in complex with its selective inhibitor BMS‐509744 and the broad‐spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL‐2‐inducible T cell kinase in the helix C‐in conformation by inducing side chain conformational changes in the ATP‐binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad‐spectrum kinase activity. BMS‐509744 uniquely stabilizes the activation loop in a substrate‐blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL‐2‐inducible T cell kinase activity. The observed BMS‐509744 binding mode allows rationalization of structure–activity relationships reported for this inhibitor class and facilitates further structure‐based drug design. Sequence‐based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.

The crystal structure of JNK2 reveals conformational flexibility in the MAP kinase insert and indicates its involvement in the regulation of catalytic activity.

c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called MAP kinase insert, which, for ERK2, has been shown to interact with regulatory proteins and, for p38alpha, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners.

X-ray crystal structure of JNK2 complexed with the p38α inhibitor BIRB796: Insights into the rational design of DFG-out binding MAP kinase inhibitors

JNK2 and p38alpha are closely related mitogen-activated protein kinases that regulate various cellular activities and are considered drug targets for inflammatory diseases. We have determined the X-ray crystal structure of the clinical phase II p38alpha inhibitor BIRB796 bound to its off-target JNK2. This shows for the first time a JNK subfamily member in the DFG-out conformation. The fully resolved activation loop reveals that BIRB796 inhibits JNK2 activation by stabilizing the loop in a position that does not allow its phosphorylation by upstream kinases. The structure suggests that substituents at the BIRB796 morpholino group and modifications of the t-butyl moiety should further increase the p38alpha to JNK2 potency ratio. For the design of selective DFG-out binding JNK2 inhibitors, the binding pocket of the BIRB796 tolyl group may have the best potential.