Targeted Nanocarriers for Systemic Delivery of IRAK4 Inhibitors to Inflamed Tissues.

Persistent and uncontrolled inflammation is the root cause of various debilitating diseases. Given that interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical modulator of inflammation, inhibition of its activity with selective drug molecules (IRAK4 inhibitors) represents a promising therapeutic strategy for inflammatory disorders. To exploit the full potential of this treatment approach, drug carriers for efficient delivery of IRAK4 inhibitors to inflamed tissues are essential. Herein, the first nanoparticle-based platform for the targeted systemic delivery of a clinically tested IRAK4 inhibitor, PF-06650833, with limited aqueous solubility (57 µg mL-1 ) is presented. The developed nanocarriers increase the intrinsic aqueous dispersibility of this IRAK4 inhibitor by 40 times. A targeting peptide on the surface of nanocarriers significantly enhances their accumulation after intravenous injection in inflamed tissues of mice with induced paw edema and ulcerative colitis when compared to non-targeted counterparts. The delivered IRAK4 inhibitor markedly abates inflammation and dramatically suppresses paw edema, mitigates colitis symptoms, and reduces proinflammatory cytokine levels in the affected tissues. Importantly, repeated injections of IRAK4 inhibitor-loaded nanocarriers have no acute toxic effect on major organs of mice. Therefore, the developed nanocarriers have the potential to significantly improve the therapeutic efficacy of IRAK4 inhibitors for different inflammatory diseases.

Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

Deciphering the intrinsic dynamics of unphosphorylated IRAK4 kinase bound to type I and type II inhibitors.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a vital protein involved in Toll-like and interleukin-1 receptor signal transduction. Several studies have reported regarding the crystal structure, dynamic properties, and interactions with inhibitors of the phosphorylated form of IRAK4. However, no dynamic properties of inhibitor-bound unphosphorylated IRAK4 have been previously studied. Herein, we report the intrinsic dynamics of unphosphorylated IRAK4 (uIRAK4) bound to type I and type II inhibitors. The corresponding apo and inhibitor-bound forms of uIRAK4 were subjected to three independent simulations of 500 ns (total 1.5 µs) each, and their trajectories were analyzed. The results indicated that all three systems were relatively stable, except for the type II inhibitor-bound form of uIRAK4, which exhibited less compact folding and higher solvent surface area. The intra-hydrogen bonds corroborated the structural deformation of the type-II inhibitor-bound complex, which could be attributed to the long molecular structure of the type-II inhibitor. Moreover, the type II inhibitor bound to uIRAK4 showed higher binding free energy with uIRAK4 than the type I inhibitor. The free energy landscape analysis showed a reorientation of Phe330 side chain from the DFG motif at different metastable states for all the systems. The intra-residual distance between residues Lys213, Glu233, Tyr262, and Phe330 suggests a functional interplay when the inhibitors are bound to uIRAK4, thereby hinting at their crucial role in the inhibition mechanism. Ultimately, the intrinsic dynamics study observed between type I/II inhibitor-bound forms of uIRAK4 may assist in better understanding the enzyme and designing therapeutic compounds.

Minimal structure of IRAK-1 to induce degradation of TRAF6.

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1-102), a serine/proline/threonine-rich ProST domain (amino acids 103-197), a central kinase domain with an activation loop (amino acids 198-522), and the C-terminal C1 and C2 domains (amino acids 523-712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585-591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants-including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.

Mechanism of MyD88S mediated signal termination.

BACKGROUND: A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)-the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited. METHODS: Luciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD). RESULTS: We show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation). CONCLUSIONS: Our results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction. Video abstract.

Pseudokinases repurpose flexibility signatures associated with the protein kinase fold for noncatalytic roles.

The bilobal protein kinase-like fold in pseudokinases lack one or more catalytic residues, conserved in canonical protein kinases, and are considered enzymatically deficient. Tertiary structures of pseudokinases reveal that their loops topologically equivalent to activation segments of kinases adopt contracted configurations, which is typically extended in active conformation of kinases. Herein, anisotropic network model based normal mode analysis (NMA) was conducted on 51 active conformation structures of protein kinases and 26 crystal structures of pseudokinases. Our observations indicate that although backbone fluctuation profiles are similar for individual kinase-pseudokinase families, low intensity mean square fluctuations in pseudo-activation segment and other sub-structures impart rigidity to pseudokinases. Analyses of collective motions from functional modes reveal that pseudokinases, compared to active kinases, undergo distinct conformational transitions using the same structural fold. All-atom NMA of protein kinase-pseudokinase pairs from each family, sharing high amino acid sequence identities, yielded distinct community clusters, partitioned by residues exhibiting highly correlated fluctuations. It appears that atomic fluctuations from equivalent activation segments guide community membership and network topologies for respective kinase and pseudokinase. Our findings indicate that such adaptations in backbone and side-chain fluctuations render pseudokinases competent for catalysis-independent roles.

Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Dimeric Structure of the Pseudokinase IRAK3 Suggests an Allosteric Mechanism for Negative Regulation.

Interleukin-1 receptor associated kinases (IRAKs) are key players in innate immune signaling that mediate the host response to pathogens. In contrast to the active kinases IRAK1 and IRAK4, IRAK2 and IRAK3 are pseudokinases lacking catalytic activity and their functions are poorly understood. IRAK3 is thought to be a negative regulator of innate immune signaling and mutations in IRAK3 are associated with asthma and cancer. Here, we report the crystal structure of the human IRAK3 pseudokinase domain in a closed, pseudoactive conformation. IRAK3 dimerizes in a unique way through a head-to-head arrangement not observed in any other kinases. Multiple conserved cysteine residues imply a potential redox control of IRAK3 conformation and dimerization. By analyzing asthma-associated mutations, we identify an evolutionarily conserved surface on IRAK3 that could form an interaction interface with IRAK4, suggesting a model for the negative regulation of IRAK4 by IRAK3.

Genome-wide identification, characterization and expression analysis of non-RD receptor like kinase gene family under Colletotrichum truncatum stress conditions in hot pepper.

Receptor like kinases (RLKs) are preserved upstream signaling molecules which regulate several biological processes from plant development to various stress adaptation programs. Non arginine aspartate (non-RD) a prominent class of RLKs plays a significant role in disease resistance and apoptosis in plants. In present investigation, a comprehensive in silico analysis for non-RD Kinase gene family as well as identification of gene structures, sequence similarity, chromosomal localization, gene duplication analysis, promoter analysis, transcript expression profiles and phylogenic studies were done. In this study, twenty-six genes were observed on nine out of twelve chromosomes. All these genes were clustered into five subfamilies under large monophyletic group termed as Interleukin-1 Receptor-Associated Kinase (IRAK) family. Some of the important physiochemical properties of twenty-six proteins are determined and ranged in the following order: (a) Amino acids size ranged from (620 to 1781) (b) Molecular weight ranged as of (70.11 to 197.11 KDa) and (c) Theoretical PI ranged from (5.69 to 8.63) respectively. Structural diversity in genomic structure among non-RD kinase gene family was identified and presence of pathogen induced cis regulatory elements including STRE, MYC, MYB, and W box were found. Expression profiles revealed the potential ability of three genes CaRLK1 from LRRXII and CaRLK15,16 from stress antifung subfamily were pointedly upregulated beyond the severe stress time period (9 DAI) in anthracnose resistant genotype PBC-80 in response to Colletotrichum truncatum infection. Subsequently, in silico studies from the available genome sequencing data helped us to identify candidate genes tangled in inducing disease resistance.

Molecular features of interleukin-1 receptor-associated kinase-b and a in Mytilus coruscus, regulating their function by infection of aquatic pathogens and the expression of their serine/threonine protein kinase functional domains.

Interleukin-1 receptor-associated kinases (IRAKs) play important roles in the innate immune system of TLR (Toll-like receptor) signaling pathway. In this paper, interleukin-1 receptor-associated kinase-b (designated as McIRAK-b) and interleukin-1 receptor-associated kinase-a (named as McIRAK-a) were obtained based on the transcriptome data, the full length of McIRAK-b was 1815 bp and McIRAK-a was 3168bp, encoding 532 and 978 amino acids, respectively. BLASTp analysis and phylogenetic relationship strongly suggested that the deduced amino acid sequence of McIRAK-b had high homology with IRAK-4 and McIRAK-a was similar to IRAK-1 of other mollusks, especially at their function domains. The expressions of McIRAK-b and McIRAK-a were detected in six tissues including adductor muscle, hemocyte, gills, gonad and hepatopancreas, and the highest expressions appeared both in gills. The expressions of McIRAK-b and McIRAK-a in gills were observed with time-dependent manners after bacterial infections. After being challenged with Vibrio alginolyticus, McIRAK-b expressed significantly and got the peak at 8 h (9.47 times compared with the control group), but the peak appeared at 4 h by being infected with Vibrio parahaemolyticus (12.02 times compared with the control group). The highest point of McIRAK-a mRNA appeared at 12 h (5.16 times) after being challenged with V.alginolyticus and 8 h (4.21 times) for V.parahaemolyticus challenge. The results suggested that IRAK-b and IRAK-a might be important in immune signaling pathway of mussels. The kinase functional domain sequences (S_TKc) of McIRAK-b and McIRAK-a expressed in BL21(DE3) and purified by Ni-NAT Superflow resin conforming to the expected molecular weight with many active sites for their conferring protein-protein interaction functions. This study may provide some further understandings of the regulatory mechanisms in the bivalve innate immune system for IRAKs family.

MyD88 Death-Domain Oligomerization Determines Myddosome Architecture: Implications for Toll-like Receptor Signaling.

Toll-like receptors (TLRs) are pivotal in triggering the innate immune response to pathogen infection. Ligand binding induces receptor dimerization which facilitates the recruitment of other post-receptor signal transducers into a complex signalosome, the Myddosome. Central to this process is Myeloid differentiation primary response 88 (MyD88), which is required by almost all TLRs, and signaling is thought to proceed via the stepwise, sequential assembly of individual components. Here, we show that the death domains of human MyD88 spontaneously and reversibly associate to form helical filaments in vitro. A 3.1-Å cryoelectron microscopy structure reveals that the architecture of the filament is identical to that of the 6:4 MyD88-IRAK4-IRAK2 hetero-oligomeric Myddosome. Additionally, the death domain of IRAK4 interacts with the filaments to reconstitute the non-stoichiometric 6:4 MyD88-IRAK4 complex. Together, these data suggest that intracellularly, the MyD88 scaffold may be pre-formed and poised for recruitment of IRAKs on receptor activation and TIR engagement.

Molecular characterization and expressional modulation of IRAK1 as downstream signaling adaptor molecule of TLR-signaling pathways in Labeo rohita following PAMPs stimulation and bacterial infections.

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19-94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338-352 aa; NEK2 (NIMA-related kinase 2) at 47-61 aa; NEK6 (NIMA-related kinase 6) at 581-595 aa and AMPK (AMP- activated protein kinase) motif at 518-538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults.

IRAK3 modulates downstream innate immune signalling through its guanylate cyclase activity.

Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3's ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade.

Discovery of a Series of 5-Azaquinazolines as Orally Efficacious IRAK4 Inhibitors Targeting MyD88L265P Mutant Diffuse Large B Cell Lymphoma.

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.

Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus).

IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate immunity, can initiate a cascade of signaling events and lead to induction of inflammatory target gene expression eventually. In the present study, we cloned and characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were highly conserved among different species. Phylogenetic tree analysis revealed that CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis indicated that CiIRAK4 was widely expressed in all tested tissues. It was significantly up-regulated after treatment with poly I:C, especially obvious in liver and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells. Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm. RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4 might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus, indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase regulated IRF5 activity through indirect ways.

Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor-associated kinase 4 (IRAK4).

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structures are in the active conformation, precluding a good understanding of IRAK4's conformational dynamics. To address this issue, here we first solved three crystal structures of the IRAK4 kinase domain (at ≤2.6 Å resolution), in its unphosphorylated, inactive state bound to either the ATP analog AMP-PNP or to one of the two small-molecule inhibitors JH-I-25 and JH-I-17. The structures disclosed that although the structure in complex with AMP-PNP is in an "αC-out" inactive conformation, those in complex with type I inhibitors assume an active "Asp-Phe-Gly (DFG)-in" and "αC-in" conformation. The ability of unphosphorylated IRAK4 to take on variable conformations prompted us to screen for small-molecule inhibitors that bind preferentially to unphosphorylated IRAK4, leading to the identification of ponatinib and HG-12-6. Solving the structures of unphosphorylated IRAK4 in complex with these two inhibitors, we found that they both bind as type II inhibitors with IRAK4 in a "DFG-out" conformation. Collectively, these structures reveal conformational flexibility of unphosphorylated IRAK4 and provide unexpected insights into the potential use of small molecules to modulate IRAK4 activity in cancer, autoimmunity, and inflammation.

A novel interleukin-1 receptor-associated kinase-4 from thick shell mussel Mytilus coruscus is involved in inflammatory response.

Interleukin-1 receptor-associated kinase-4 (IRAK4) is considered as the most upstream kinase of IRAKs and plays a vital role in Toll-like receptor/Interleukin-1 receptor (TLR/IL-1R) signal transduction. In the present study, IRAK4 from thick shell mussel Mytilus coruscus (McIRAK4) was identified and characterized. McIRAK4 showed the most similarity to its counterparts in bivalves. The conserved death domain (DD) and catalytic domain of serine/threonine kinases (STKc) were predicted in all examined IRAK4s. McIRAK4 transcripts were constitutively expressed in all examined tissues with the higher expression level in immune related tissues, and were significantly induced in haemocytes upon lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further, the expression of McIRAK4 was obviously repressed by dsRNA mediated RNA interference (RNAi), meanwhile the proinflammatory cytokines TNF-alpha and IL17 were down-regulated while the antiinflammatory cytokine TGF-ß was up-regulated. Additionally, McIRAK4 showed a global cytoplasmic localization in HEK293T cells through fluorescence microscopy. These results collectively indicated that McIRAK4 is one member of IRAK4 subfamily and might play the potential signal transducer role in inflammatory response. The present study provides supplement for TLR-mediated signaling pathway triggered by pathogenic invasions in thick shell mussel, and contributes to the clarification of the innate immune response in molluscs.

Identification, characterisation and preliminary functional analysis of IRAK-M in grass carp (Ctenopharyngodon idella).

Interleukin-1 receptor-associated kinase (IRAK) family members play important roles in myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) signaling, the crucial innate immune pathway in vertebrates. In the present study, the IRAK family gene IRAK-M (also called IRAK3) from grass carp (Ctenopharyngodon idella) was cloned and characterised. IRAK-M was mainly enriched in the spleen, and the significantly altered expression was observed after grass carp reovirus (GCRV) infection. Subcellular localisation showed that IRAK-M protein distributed uniformly in the entire cell and co-localised with MyD88 in the cytoplasm of transfected cells. Additionally, the interaction between IRAK-M and MyD88 was confirmed by bimolecular fluorescence complementation (BiFC) system. Moreover, deficient of IRAK-M in C. idella kidney cell line (CIK) with small interference RNA (siRNA) upregulated polyinosinic:polycytidylic acid (poly(I:C))-induced inflammatory cytokines production, including interleukin 8 (IL-8), IL-6, and tumour necrosis factor α (TNF-α), which reveals that IRAK-M functions as a negative regulator of inflammatory cytokines. Taken together, our results demonstrate that IRAK-M gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the IRAK-M in teleosts.

Assessing the risk of drug crystallization in vivo.

INTRODUCTION: Low intrinsic solubility leading to poor oral bioavailability is a common challenge in drug discovery that can often be overcome by formulation strategies, however, it remains a potential limitation that can pose challenges for early risk assessment and represent a significant obstacle to drug development. We identified a selective inhibitor (BMS-986126) of the IL-1 receptor-associated kinase 4 (IRAK4) with favorable properties as a lead candidate, but with unusually low intrinsic solubility of <1 µg/mL. METHODS: Conventional histopathology identified the issue of crystal formation in vivo. Subsequent investigative work included confocal Raman micro-spectroscopy, MALDI-MS, polarized light microscopy of fresh wet-mount tissue scrapings and transmission electron microscopy. RESULTS: BMS-986126 was advanced into a 2-week toxicology study in rats. The main finding in this study was minimal granulomatous inflammation in the duodenum, associated with the presence of birefringent crystals at the highest dosage of 100 mg/kg/day. Considering the safety margin, and the single location of the lesion, BMS-986126 was further progressed into IND-enabling toxicology studies where tolerability deteriorated with increasing dosing duration. Birefringent crystals and granulomatous inflammation were detected in multiple organs at dosages ≥20 mg/kg/day. Raman spectroscopy confirmed the identity of the crystals as BMS-986126. Therefore, follow up investigations were conducted to further characterize drug crystallization and to evaluate detection methods for their potential to reliably detect in vivo crystallization early. DISCUSSION: The purpose of our efforts was to identify critical factors influencing in vivo drug crystallization and to provide a preliminary assessment (based on one compound) which method would be best suited for identifying crystals. Results indicated a combination of methods was required to provide a complete assessment of drug crystallization and that a simple technique, scraping of freshly collected tissue followed by evaluation under polarizing light was suitable for detecting crystals. However, dosing for 2 weeks was required for crystals to grow to a clearly detectable size.