Recent Advances in IRAK1: Pharmacological and Therapeutic Aspects.

Interleukin receptor-associated kinase (IRAK) proteins are pivotal in interleukin-1 and Toll-like receptor-mediated signaling pathways. They play essential roles in innate immunity and inflammation. This review analyzes and discusses the physiological functions of IRAK1 and its associated diseases. IRAK1 is involved in a wide range of diseases such as dry eye, which highlights its potential as a therapeutic target under various conditions. Various IRAK1 inhibitors, including Pacritinib and Rosoxacin, show therapeutic potential against malignancies and inflammatory diseases. The covalent IRAK1 inhibitor JH-X-119-01 shows promise in B-cell lymphomas, emphasizing the significance of covalent bonds in its activity. Additionally, the emergence of selective IRAK1 degraders, such as JNJ-101, provides a novel strategy by targeting the scaffolding function of IRAK1. Thus, the evolving landscape of IRAK1-targeted approaches provides promising avenues for increasingly safe and effective therapeutic interventions for various diseases.

Effect of Divalent Metal Ions on the Ribonuclease Activity of the Toxin Molecule HP0894 from Helicobacter pylori.

Bacteria and archaea respond and adapt to environmental stress conditions by modulating the toxin-antitoxin (TA) system for survival. Within the bacterium Helicobacter pylori, the protein HP0894 is a key player in the HP0894-HP0895 TA system, in which HP0894 serves as a toxin and HP0895 as an antitoxin. HP0894 has intrinsic ribonuclease (RNase) activity that regulates gene expression and translation, significantly influencing bacterial physiology and survival. This activity is influenced by the presence of metal ions such as Mg2+. In this study, we explore the metal-dependent RNase activity of HP0894. Surprisingly, all tested metal ions lead to a reduction in RNase activity, with zinc ions (Zn2+) causing the most significant decrease. The secondary structure of HP0894 remained largely unaffected by Zn2+ binding, whereas structural rigidity was notably increased, as revealed using CD analysis. NMR characterized the Zn2+ binding, implicating numerous His, Asp, and Glu residues in HP0894. In summary, these results suggest that metal ions play a regulatory role in the RNase activity of HP0894, contributing to maintaining the toxin molecule in an inactive state under normal conditions.

Structural Deformation of MTX Induced by Nanodrug Conjugation Dictate Intracellular Drug Transport and Drug Efficacy.

BACKGROUND: Understanding structural interactions between the active drug and conjugated nanoparticles is critical for optimizing intracellular drug transport and for increasing nano drug efficacy. In this regard, analyzing the conformational deformation of conjugated drugs surrounding nanoparticles is essential to understand the corresponding nanodrug efficacy. PURPOSE: The objective of this study is to present an optimal synthesis method for efficient drug delivery through a clear structural analysis of nanodrugs according to the type of conjugation. METHODS AND RESULTS: In this study, the structural variation of methotrexate (MTX) surrounding carbon nanotubes, depending on the type of conjugation style, such as covalent and non-covalent (PEGylation) bonds, was investigated. Specifically, covalent bonds of MTX surrounding CNTs induced greater structural deformation compared to non-covalent bonds (ie, PEGylated CNT). CONCLUSION: Greater changes in the structural variations of MTX analyzed by nuclear magnetic resonance (NMR) significantly improved the anti-inflammatory drug efficacy of human fibroblast-like synovial cells (FLS) via stable drug release in the extracellular environment and burst drug release under intracellular conditions.

Structural ensemble-based docking simulation and biophysical studies discovered new inhibitors of Hsp90 N-terminal domain.

Heat shock protein 90 (Hsp90) is one of the most abundant cellular proteins and plays a substantial role in the folding of client proteins. The inhibition of Hsp90 has been regarded as an attractive therapeutic strategy for treating cancer because many oncogenic kinases are Hsp90 client proteins. In this study, we report new inhibitors that directly bind to N-terminal ATP-binding pocket of Hsp90. Optimized structure-based virtual screening predicted candidate molecules, which was followed by confirmation using biophysical and cell-based assays. Among the reported crystal structures, we chose the two structures that show the most favourable early enrichments of true-positives in the receiver operating characteristic curve. Four molecules showed significant changes in the signals of 2D [1H, 15N] correlation NMR spectroscopy. Differential scanning calorimetry analysis supported the results indicating direct binding. Quantified dissociation constant values of the molecules, determined by a series of 2D NMR experiments, lie in the range of 0.1-33 µM. Growth inhibition assay with breast and lung cancer cells confirmed the cellular activities of the molecules. Cheminformatics revealed that the molecules share limited chemical similarities with known inhibitors. Molecular dynamics simulations detailed the putative binding modes of the inhibitors.

Metabolomic profiling of CSF in multiple sclerosis and neuromyelitis optica spectrum disorder by nuclear magnetic resonance.

Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are inflammatory diseases of the central nervous system. Although several studies have characterized the metabolome in the cerebrospinal fluid (CSF) from MS and NMOSD patients, comparative analyses between them and between the relapse and the remission of each disease have not been performed. Both univariate and multivariate analyses were used to compare 1H-NMR spectra of CSF from MS, NMOSD, and healthy controls (HCs). The statistical analysis showed alterations of eight metabolites that were dependent on the disease. Levels of 2-hydroxybutyrate, acetone, formate, and pyroglutamate were higher and levels of acetate and glucose were lower in both MS and NMOSD. Citrate was lower in MS patients, whereas lactate was higher in only NMOSD specifically. The shared feature of metabolic changes between MS and NMOSD may be related to altered energy metabolism and fatty acid biosynthesis in the brain. Another analysis to characterize relapse and remission status showed that isoleucine and valine were down-regulated in MS relapse compared to MS remission. The other metabolites identified in the disease comparison showed the same alterations regardless of disease activity. These findings would be helpful in understanding the biological background of these diseases, and distinguishing between MS and NMOSD, as well as determining the disease activity.

The Smad7-Skp2 complex orchestrates Myc stability, impacting on the cytostatic effect of TGF-ß.

In most human cancers the Myc proto-oncogene is highly activated. Dysregulation of Myc oncoprotein contributes to tumorigenesis in numerous tissues and organs. Thus, targeting Myc stability could be a crucial step for cancer therapy. Here we report Smad7 as a key molecule regulating Myc stability and activity by recruiting the F-box protein, Skp2. Ectopic expression of Smad7 downregulated the protein level of Myc without affecting the transcription level, and significantly repressed its transcriptional activity, leading to inhibition of cell proliferation and tumorigenic activity. Furthermore, Smad7 enhanced ubiquitylation of Myc through direct interaction with Myc and recruitment of Skp2. Ablation of Smad7 resulted in less sensitivity to the growth inhibitory effect of TGF-ß by inducing stable Myc expression. In conclusion, these findings that Smad7 functions in Myc oncoprotein degradation and enhances the cytostatic effect of TGF-ß signaling provide a possible new therapeutic approach for cancer treatment.

DRAK2 participates in a negative feedback loop to control TGF-ß/Smads signaling by binding to type I TGF-ß receptor.

TGF-ß1 is a multifunctional cytokine that mediates diverse biological processes. However, the mechanisms by which the intracellular signals of TGF-ß1 are terminated are not well understood. Here, we demonstrate that DRAK2 serves as a TGF-ß1-inducible antagonist of TGF-ß signaling. TGF-ß1 stimulation rapidly induces DRAK2 expression and enhances endogenous interaction of the type I TGF-ß receptor with DRAK2, thereby blocking R-Smads recruitment. Depletion of DRAK2 expression markedly augmented the intensity and the extent of TGF-ß1 responses. Furthermore, a high level of DRAK2 expression was observed in basal-like and HER2-enriched breast tumors and cell lines, and depletion of DRAK2 expression suppressed the tumorigenic ability of breast cancer cells. Thus, these studies define a function for DRAK2 as an intrinsic intracellular antagonist participating in the negative feedback loop to control TGF-ß1 responses, and aberrant expression of DRAK2 increases tumorigenic potential, in part, through the inhibition of TGF-ß1 tumor suppressor activity.

Suppressive effect of an isoflavone fraction on tumor necrosis factor-alpha-induced interleukin-8 production in human intestinal epithelial Caco-2 cells.

This study demonstrates the effect of soybean components on the tumor necrosis factor-alpha (TNF-alpha)-induced production of interleukin-8 (IL-8), one of the major inflammatory chemokines, in intestinal epithelial-like Caco-2 cells. Among the soybean components, an isoflavone fraction (IFF) suppressed the TNF-alpha-induced IL-8 secretion by Caco-2 cells in a dose-dependent manner, whereas a soyasaponin fraction and soypeptide fraction had no significant effect on TNF-alpha-induced IL-8 secretion. The IL-8 secretion induced by hydrogen peroxide and by IL-1beta was not suppressed by IFF, suggesting that the inhibitory effect of isoflavone was specific for the TNF-alpha-induced regulation of IL-8. The increased expression of IL-8 mRNA by TNF-alpha was almost completely suppressed by IFF. Furthermore, the transcriptional activity of the human IL-8 promoter was increased by the TNF-alpha treatment, and IFF significantly suppressed the IL-8 promoter activity. These results indicate that IFF suppressed TNF-alpha-induced IL-8 production at the transcriptional level in human intestinal Caco-2 cells, suggesting IFF of soybean as a promising food component for preventing intestinal inflammation such as inflammatory bowel disease.

Cycloheximide treatment induces the uptake of neutral and dibasic amino acids via the activation of system b(0,+) in human intestinal Caco-2 cells.

Amino acids in enterocytes are thought to be absorbed in the intestinal epithelium via various types of amino acid transport, although the regulation of these amino acid transport systems has not been elucidated. We examined in the present study the effect of several inhibitors involved in mRNA and protein synthesis, and of protein translocation on the L-leucine (Leu) uptake in human intestinal epithelial-like Caco-2 cells. Culturing Caco-2 cells with cycloheximide (CHX) enabled the L-Leu uptake to be significantly increased in a dose- and time-dependent manner. The uptake of L-lysine (Lys) was also increased by the CHX treatment, whereas the uptake of L-glutamate, taurine, and Gly-Gln was not changed. Among the two transport systems, b(0,+) and y+, which are known to be involved in L-Lys uptake by Caco-2, the system b(0,+) component was greatly increased by the CHX treatment, suggesting that system b(0,+) was mainly responsible for the increase in L-Leu and L-Lys uptake. The mRNA levels of rBAT and b(0,+) AT, whose molecules comprise system b(0,+), were both significantly increased by the CHX treatment in a time-dependent manner. These results strongly suggest that the CHX treatment increased the Leu and Lys uptake by activating system b(0,+) and inducing rBAT and b(0,+) AT mRNA expression in human intestinal epithelial Caco-2 cells.

Cadmium induces interleukin-8 production via NF-kappaB activation in the human intestinal epithelial cell, Caco-2.

In the present study, we investigated the effect of CdCl2 on the inflammatory cytokines in human intestinal Caco-2 cells. The secretion of IL-8 from Caco-2 cells was significantly increased in a dose- and time-dependent manner, whereas the secretion of such other inflammatory cytokines as TNF-alpha and IFN-gamma was not changed. And IL-8 mRNA level was significantly increased by exposing the cells to CdCl2. A reporter vector containing the IL-8 promoter region was then constructed to determine the IL-8 transcriptional activity. The results of this assay demonstrated that the transcriptional activity of IL-8 was increased by CdCl2. Treatment with PDTC, an NF-kappaB inhibitor, suppressed the IL-8 secretion in Caco-2 cells. Site-directed mutation of the NF-kappaB consensus element in the human IL-8 promoter abolished the increased transcriptional activity by CdCl2. The increased transcriptional activity caused by CdCl2 was also suppressed in an NF-kappaB knock-down Caco-2 cell line that had been stably established by the RNAi method. The increase in translocation of the NF-kappaB protein into the nucleus and I-kappaB alpha degradation resulting from CdCl2 stimulation was also confirmed by a Western analysis. Our results suggest that CdCl2 induced IL-8 secretion, its transcription, and its transcriptional activation regulated by NF-kappaB via I-kappaB alpha degradation.

Oral exposure to cadmium chloride triggers an acute inflammatory response in the intestines of mice, initiated by the over-expression of tissue macrophage inflammatory protein-2 mRNA.

Intestinal inflammation is an indispensable protective response of the gut immune system to aggressive injury from pathogens and/or chemicals. Although the major route of exposure to cadmium for most people is via food, causing the gastrointestinal tract to become the first target organ, very little information is available on whether cadmium exposure triggers the intestinal inflammatory response. We investigated in the present study the acute inflammatory response in the intestines of mice orally challenged with a single dose of cadmium chloride (CdCl(2)) by determining the gene expression of pro-inflammatory mediators with real-time PCR, and by examining the infiltration of inflammatory cells with a myeloperoxidase (MPO) assay and histological analysis of hematoxylin and eosin (H&E)-stained intestinal sections. The results show that CdCl(2) significantly increased the expression of macrophage inflammatory protein-2 mRNA (30-40 times the normal level) 3h and the activity of MPO (about 2 times the normal level) 24h after the challenge in the duodenal and proximal jejunal tissue. Furthermore, these increases were dose-dependent over a dosage range of 25-100mg/kg of body weight. The histological analysis confirmed that CdCl(2) induced mild to moderate villus damage and infiltration of inflammatory cells into the lamina propria. All these results demonstrate that oral exposure to CdCl(2) triggered an acute inflammatory response in the proximal intestine of mice, suggesting that the gut immune system was involved in the toxic effects of Cd on the gastrointestinal tract.