IKKβ Inhibitor Research Articles

A Novel IKKβ Inhibitor Ameliorates Podocyte and Kidney Injuries

A Novel IKKβ Inhibitor Ameliorates Podocyte and Kidney Injuries

Dehydroevodiamine targeting IKKβ to alleviate acute gastric injury via inhibiting the p65/NLRP3 axis

BackgroundAcute gastric injury, a common and recurring global digestive disorder, significantly impairs patient quality of life and overall health. Dehydroevodiamine (DHE), a bioactive natural product derived from Tetradium ruticarpum (A. Juss.) Hartley, shows potential therapeutic effects on acute gastric injury. This study investigates the underlying mechanisms of DHE's alleviating effects on acute gastric injury. MethodsThe gastric mucosal protective effect of DHE was confirmed through in vivo and in vitro acute gastric injury models. Biotin pulldown MS and molecular dynamics simulations identified DHE's target. CETSA and SPR assays validated DHE's affinity for IKKβ. Protein site mutation validation and MST pinpointed the direct binding sites of DHE on IKKβ. Additionally, the potential mechanism by which DHE ameliorates acute gastric injury was elucidated using WB, IHC, and IF methods, and further confirmed by rescue experiments. ResultsDHE effectively ameliorated IDO-induced gastric injury in GES-1 cells and rat gastric mucosa, both in vitro and in vivo. Biotin pulldown MS identified IKKβ as the target of DHE in alleviating gastric injury. CETSA and SPR assays confirmed DHE's direct binding to IKKβ. Molecular dynamics simulations, protein mutation experiments, and MST results pinpointed GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket as the binding sites of DHE on IKKβ. Notably, DHE was found to competitively bind to IKKβ with ATP. Mechanistically, DHE attenuated IDO-induced gastric injury by inhibiting the IKKβ-p65/NLRP3 signaling pathway. Importantly, exogenous activation of IKKβ reversed the therapeutic effect of DHE, indicating that DHE's efficacy depends on IKKβ. ConclusionDHE attenuated IDO-induced gastric injury by inhibiting the IKKβ-p65/NLRP3 signaling pathway. Notably, DHE is a novel ATP-competitive IKKβ inhibitor that prevents phosphorylation by targeting GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket. This study reveals new targets of action for DHE, providing a new molecular basis for using DHE in treating inflammation-related diseases.

Costunolide Inhibits Chronic Kidney Disease Development by Attenuating IKKβ/NF-κB Pathway.

Chronic kidney disease (CKD) is a significant worldwide health concern that leads to high mortality rates. The bioactive substance costunolide (CTD) has demonstrated several pharmacological effects and holds promise as a CKD treatment. This study aims to investigate the impact of CTD on CKD and delve into its mechanisms of action. Unilateral ureteral obstruction (UUO) methods and renal fibrosis mice models were created. Various concentrations of CTD were injected into UUO mice models to investigate the therapeutic effects of CTD on renal fibrosis of mice. Then, renal morphology, pathological changes, and the expression of genes related to fibrosis, inflammation and ferroptosis were analysed. RNA sequencing was utilized to identify the main biological processes and pathways involved in renal injury. Finally, both overexpression and inhibition of IKKβ were studied to examine their respective effects on fibrosis and inflammation in both in vitro and in vivo models. CTD treatment was found to significantly alleviate fibrosis, inflammation and ferroptosis in UUO-induced renal fibrosis mice models. The results of RNA sequencing suggested that the IKKβ acted as key regulatory factor in renal injury and the expression of IKKβ was increased in vitro and in vivo renal fibrosis model. Functionally, down-regulated IKKβ expression inhibits ferroptosis, inflammatory cytokine production and collagen deposition. Conversely, IKKβ overexpression exacerbates progressive renal fibrosis. Mechanistically, CTD alleviated renal fibrosis and inflammation by inhibiting the expression of IKKβ and attenuating IKKβ/NF-κB pathway. This study demonstrates that CTD could mitigate renal fibrosis, ferroptosis and inflammation in CKD by modulating the IKKβ/NF-κB pathway, which indicates targeting IKKβ has an enormous potential for treating CKD.

Dual targeting of inflammation and β-cell dysfunction for therapy of diabetes mellitus

Diabetes Mellitus (DM) is a chronic metabolic disorder characterized by β-cell loss and inflammation. From a therapeutic standpoint, it would be necessary to halt the uncontrolled inflammatory response and to enable the regeneration of the damaged β-cells. In this work, to achieve the latter two drug-loaded nanocarrier systems have been developed and evaluated in-vivo in DM mouse model. For β-cell proliferation, small chitosan (CS) nanoparticles (NPs) (201 ± 30 nm) were loaded with 4b, a selective inhibitor of DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A). To reduce inflammation, large poly lactic-co-glycolic acid (PLGA) microparticles (MPs) (1172 ± 77 nm) were encapsulated with BI-605906, an inhibitor of IKKβ (inhibitor of kappa B kinase beta), acting as an inhibitor of an inhibitor. The EE% values were 90 ± 4.2 % and 87 ± 4.46 % for 4b–CS–NPs and BI-605906-PLGA-MPs, respectively. In-vivo biodistribution studies indicated the ability of both particles to accumulate in the pancreas. For CS-NPs, this was achieved as a function of the increased vascular permeability in DM, whereas for PLGA-MPs, this was achieved as function of macrophage uptake. Treatment with 4b–CS–NPs (but not the unloaded drug) reduced blood glucose levels (BGLs) and elevated cyclin D1 (CD1) gene expression indicating the successful ability of the NP system in promoting β-cells proliferation. Although treatment with free BI-605906 showed a modest reduction in BGLs, BI-605906-PLGA-MPs showed no improvement in BGLs. The latter could possibly be attributed to sub-optimal dosing. These results indicate that 4b–CS–NPs have the potential to lower BGLs by promoting β-cell proliferation and restoring pancreatic function. Further optimization is needed for BI-605906-PLGA-MPs, which may enhance therapeutic outcome by dual therapy.

The discovery of a novel IκB kinase β inhibitor based on pharmacophore modeling, virtual screening and biological evaluation.

Background: IκB kinase β (IKKβ) plays a pivotal role in the NF-κB signaling pathway and is considered a promising therapeutic target for various diseases. Materials & methods: The authors developed and validated a 3D pharmacophore model of IKKβ inhibitors via the HypoGen algorithm in Discovery Studio 2019, then performed virtual screening, molecular docking and kinase assays to identify hit compounds from the ChemDiv database. The compound with the highest inhibitory activity was further evaluated in adjuvant-induced arthritis rat models. Results: Among the four hit compounds, Hit 4 had the highest IKKβ inhibitory activity (IC50=30.4±3.8), and it could significantly ameliorate joint inflammation and damage in vivo. Conclusion: The identified compound, Hit 4, can be optimized as a therapeutic agent for inflammatory diseases.

Inhibition of IKKβ via a DNA-Based In Situ Delivery System Improves Achilles Tendinopathy Healing in a Rat Model

Background: The inhibition of IKKβ by the inhibitor 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3-pyridine carbonitrile (ACHP) is a promising strategy for the treatment of Achilles tendinopathy. However, the poor water solubility of ACHP severely hinders its in vivo application. Moreover, the effective local delivery of ACHP to the tendon and its therapeutic effects have not been reported. Purpose: To investigate the therapeutic effects of IKKβ inhibition via injection of ACHP incorporated into a DNA supramolecular hydrogel in a collagenase-induced tendinopathy rat model. Study Design: Controlled laboratory study. Methods: Dendritic DNA, a Y-shaped monomer, and a crosslinking monomer were mixed with ACHP and self-assembled into an ACHP-DNA supramolecular hydrogel (ACHP-Gel). The effects of ACHP-Gel in tendon stem/progenitor cells were investigated via RNA sequencing and validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A total of 120 collagenase-induced rats were randomly assigned to 5 groups: blank, phosphate-buffered saline (PBS), DNA-Gel, ACHP, and ACHP-Gel. Healing outcomes were evaluated using biomechanic and histologic evaluations at 4 and 8 weeks. Results: ACHP-Gel enhanced the solubility of ACHP and sustained its release for ≥21 days in vivo, which significantly increased the retention time of ACHP and markedly reduced the frequency of administration. RNA sequencing and qRT-PCR showed that ACHP effectively downregulated genes related to inflammation and extracellular matrix remodeling and upregulated genes related to tenogenic differentiation. The cross-sectional area (P = .024), load to failure (P = .002), stiffness (P = .039), and elastic modulus (P = .048) significantly differed between the ACHP-Gel and PBS groups at 8 weeks. The ACHP-Gel group had better histologic scores than the ACHP group at 4 (P = .042) and 8 weeks (P = .009). Type I collagen expression (COL-I; P = .034) and the COL-I/collagen type III ratio (P = .015) increased while interleukin 6 expression decreased (P < .001) in the ACHP-Gel group compared with the ACHP group at 8 weeks. Conclusion: DNA supramolecular hydrogel significantly enhanced the aqueous solubility of ACHP and increased its release-retention time. Injection frequency was markedly reduced. ACHP-Gel suppressed inflammation in Achilles tendinopathy and promoted tendon healing in a rat model. Clinical Relevance: ACHP-Gel injection is a promising strategy for the treatment of Achilles tendinopathy in clinical practice.

The effects of NF-κB suppression on the early healing response following intrasynovial tendon repair in a canine model.

The highly variable clinical outcomes noted after intrasynovial tendon repair have been associated with an early inflammatory response leading to the development of fibrovascular adhesions. Prior efforts to broadly suppress this inflammatory response have been largely unsuccessful. Recent studies have shown that selective inhibition ofIkappaB kinase beta(IKK-β), an upstream activator ofnuclear factor kappa-light chain enhancer of activated B cells(NF-κB) signaling, mitigates the early inflammatory response and leads to improved tendon healing outcomes. In the current study, we test the hypothesis that oral treatment with the IKK-β inhibitor ACHP (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinenitrile an inhibitor) will modulate the postoperative inflammatory response and improve intrasynovial flexor tendon healing. To test this hypothesis, the flexor digitorum profundus tendon of 21 canines was transected and repaired within the intrasynovial region and assessed after 3 and 14 days. Histomorphometry, gene expression analyses, immunohistochemistry, and quantitative polarized light imagingwere used to examine ACHP-mediated changes. ACHP led to reduction in phosphorylated p-65, indicating that NF-κB activity was suppressed. ACHP enhanced expression of inflammation-related genes at 3 days and suppressed expression of these genes at 14 days. Histomorphometry revealed enhanced cellular proliferation and neovascularization in ACHP-treated tendons compared with time-matched controls. These findings demonstrate that ACHP effectively suppressed NF-κB signaling and modulated early inflammation, leading to increased cellular proliferation and neovascularization without stimulating the formation of fibrovascular adhesions. Together, these data suggest that ACHP treatment accelerated the inflammatory and proliferative phases of tendon healing following intrasynovial flexor tendon repair. Clinical Significance: Using a clinically relevant large-animal model, this study revealed that targeted inhibition of nuclear factor kappa-light chain enhancer of activated B cellssignaling with ACHP provides a new therapeutic strategy for enhancing the repair of sutured intrasynovial tendons.

IKKβ Inhibition Attenuates Epithelial Mesenchymal Transition of Human Stem Cell-Derived Retinal Pigment Epithelium.

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-β) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF-β/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE-EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

Targeting IKKβ Activity to Limit Sterile Inflammation in Acetaminophen-Induced Hepatotoxicity in Mice.

The kinase activity of inhibitory κB kinase β (IKKβ) acts as a signal transducer in the activating pathway of nuclear factor-κB (NF-κB), a master regulator of inflammation and cell death in the development of numerous hepatocellular injuries. However, the importance of IKKβ activity on acetaminophen (APAP)-induced hepatotoxicity remains to be defined. Here, a derivative of caffeic acid benzylamide (CABA) inhibited the kinase activity of IKKβ, as did IMD-0354 and sulfasalazine which show therapeutic efficacy against inflammatory diseases through a common mechanism: inhibiting IKKβ activity. To understand the importance of IKKβ activity in sterile inflammation during hepatotoxicity, C57BL/6 mice were treated with CABA, IMD-0354, or sulfasalazine after APAP overdose. These small-molecule inhibitors of IKKβ activity protected the APAP-challenged mice from necrotic injury around the centrilobular zone in the liver, and rescued the mice from hepatic damage-associated lethality. From a molecular perspective, IKKβ inhibitors directly interrupted sterile inflammation in the Kupffer cells of APAP-challenged mice, such as damage-associated molecular pattern (DAMP)-induced activation of NF-κB activity via IKKβ, and NF-κB-regulated expression of cytokines and chemokines. However, CABA did not affect the upstream pathogenic events, including oxidative stress with glutathione depletion in hepatocytes after APAP overdose. N-acetyl cysteine (NAC), the only FDA-approved antidote against APAP overdose, replenishes cellular levels of glutathione, but its limited efficacy is concerning in late-presenting patients who have already undergone oxidative stress in the liver. Taken together, we propose a novel hypothesis that chemical inhibition of IKKβ activity in sterile inflammation could mitigate APAP-induced hepatotoxicity in mice, and have the potential to complement NAC treatment in APAP overdoses.

ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm.

Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation and vascular inflammation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation. Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development/dissection. Ang II (Angiotensin II) infusion of ApoE-/- mouse model combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation/dissection. The relevance of macrophage ADAR1 to human AAA was examined using human aneurysm specimens. Moreover, a novel humanized AAA model was established to test the role of human macrophages in aneurysm formation in human arteries. Allograft transplantation of wild-type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that nonvascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence and severity of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB (nuclear factor kappa B) signaling, and enhanced the expression of a number of anti-inflammatory microRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 (DiGeorge syndrome critical region 8) interaction, and consequently inhibited pri- to pre-microRNA processing of microRNAs targeting IKKβ, resulting in an increased IKKβ (inhibitor of nuclear factor kappa-B) expression and enhanced NF-κB signaling. Significantly, ADAR1 was induced in macrophages and interacted with Drosha in human AAA lesions. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism, that is, Drosha protein degradation, which inhibits the processing of microRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation in AAA.

Therapeutic Potential of Catechin as an IKK-β Inhibitor for the Management of Arthritis: In vitro and In vivo Approach.

Rheumatoid arthritis (RA) is associated with increased levels of cytokines, for instance, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interleukin-1 (IL-1), which exhibit potent pro-inflammatory effects and are contributing factors to disease progression. A range of cytokines, cell adhesion molecules, and enzymes that are implicated in the debilitating effects of RA are transcribed by nuclear factor kappa. The purpose of this research was to characterize the efficacy of "catechin" as an IkappaB kinase-beta (IKK-β) inhibitor in collagen-induced arthritis (CIA) model in mice, as IKK-β is crucial in the transmission of signal-inducible NF-κβ activation. Arthritis was brought on in Bagg and Albino, but it is written BALB/c (BALB/c) male mice through subcutaneous immunization with bovine type II collagen on days 0 and 21. Catechin is given orally every day after the onset of the disease. Clinical evaluation of the prevalence and severity of the condition was done throughout the trial, and biochemical testing was done at the end (day 42). In vitro findings of the study demonstrated catechin as a potent inhibitor of IKK-β with Half maximal Inhibitory Concentration (IC50) values of 2.90 μM and 4.358 μM in IKK-β and NF-κβ transactivation activity assay, respectively. Furthermore, catechin (dose range of 10-100 mg/kg, p.o.) was effective in reducing disease incidence and clinical signs in a dose-dependent manner, with an Effective Dose for 50% of the population (ED50) value of 79.579 mg/kg. The findings of this study demonstrate dose-dependent efficacy in terms of both disease severity (clinical scoring) and inflammatory markers (biochemical evaluation of the serum and joints). IKK inhibitors are a prospective target for the creation of new therapeutics for arthritis and other inflammatory diseases because it has been suggested that this enzyme is crucial in the pathophysiology of RA. The finding of this study suggests that "catechin" represents a novel inhibitor of IKK-β with promising anti-inflammatory activity.

IκB kinase β (IKKβ): Structure, transduction mechanism, biological function, and discovery of its inhibitors.

The effective approach to discover innovative drugs will ask natural products for answers because of their complex and changeable structures and multiple biological activities. Inhibitory kappa B kinase beta (IKKβ), known as IKK2, is a key regulatory kinase responsible for the activation of NF-κB through its phosphorylation at Ser177 and Ser181 to promote the phosphorylation of inhibitors of kappa B (IκBs), triggering their ubiquitination and degradation to active the nuclear factor kappa-B (NF-κB) cascade. Chemical inhibition of IKKβ or its genetic knockout has become an effective method to block NF-κB-mediated proliferation and migration of tumor cells and inflammatory response. In this review, we summarized the structural feature and transduction mechanism of IKKβ and the discovery of inhibitors from natural resources (e.g. sesquiterpenoids, diterpenoids, triterpenoids, flavonoids, and alkaloids) and chemical synthesis (e.g. pyrimidines, pyridines, pyrazines, quinoxalines, thiophenes, and thiazolidines). In addition, the biosynthetic pathway of novel natural IKKβ inhibitors and their biological potentials were discussed. This review will provide inspiration for the structural modification of IKKβ inhibitors based on the skeleton of natural products or chemical synthesis and further phytochemistry investigations.

Effects of tumor necrosis factor-α on glucose uptake in human granulosa cells under high androgen conditions.

Hyperandrogenism is a key pathological characteristic of polycystic ovary syndrome (PCOS). Tumor necrosis factor α (TNF-α) is both an adipokine and a chronic inflammatory factor, which has been proven to be involved in the pathologic process of PCOS. This study aimed to determine how TNF-α affects glucose uptake in human granulosa cells in the presence of high testosterone concentration. KGN cell line was treated with testosterone and TNF-α alone or co-culture combination for 24 hr, or starved for 24 hr. Quantitative real-time polymerase chain reaction (qPCR) and western blot were performed to measure glucose transporter type 4 (GLUT4) message RNA (mRNA) and protein expression in treated KGN cells. Glucose uptake and GLUT4 expression were detected by immunofluorescence (IF). Furthermore, western blot was performed to measure the contents in the nuclear factor kappa-B (NF-κB) pathway. Meantime, upon addition of TNF-α receptor II (TNFRII) inhibitor or Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) antagonist to block the TNFRII-IKKβ-NF-κB signaling pathway, the glucose uptake in KGN cells and GLUT4 translocation to cytomembrane were detected by IF, and related proteins in TNFRII-IKKβ-NF-κB were detected by western blot. The glucose uptake in Testosterone + TNF-α group was lowered significantly, and Total GLUT4 mRNA and proteins were reduced significantly. GLUT4 translocation to cytomembrane was tarnished visibly; concurrently, the phosphorylated proteins in the TNFRII-IKKβ-NF-κB signaling pathway were enhanced significantly. Furthermore, upon addition of TNFRII inhibitor or IKKβ inhibitor to block the TNFRII-IKKβ-NF-κB signaling pathway, the glucose uptake of treated granulosa cells was improved. TNFRII and IKKβ antagonists may improve glucose uptake in granulosa cells induced by TNF-α by blocking the TNFRII-IKKβ-NF-κB signaling pathway under high androgen conditions.

Avanafil mediated dual inhibition of IKKβ and TNFR1 in an experimental paradigm of Alzheimer’s disease: in silico and in vivo approach

In Alzheimer’s disease pathology, inhibitors of nuclear factor kappa-β kinase subunit β (IKKB) and Tumor necrosis factor receptor 1 (TNFR1) signaling are linked to neuroinflammation-mediated cognitive decline. We explored the role of a phosphodiesterase 5 inhibitor (PDE5I) with dual antagonistic action on IKKB and TNFR1 to inhibit nuclear factor kappa B (NF-kB) and curb neuroinflammation. In the in silico approach, the FDA-approved Zinc 15 library was docked with IKKB and TNFR1. The top compound with dual antagonistic action on IKKB and TNFR1 was selected based on bonding and non-bonding interactions. Further, induced fit docking (IFD), molecular mechanics—generalized Born and surface area (MMGBSA), and molecular dynamic studies were carried out and evaluated. Lipopolysaccharide (LPS) administration caused a neuroinflammation-mediated cognitive decline in mice. Two doses of avanafil were administered for 28 days while LPS was administered for 10 days. Morris water maze (MWM) along with the passive avoidance test (PAT) were carried out. Concurrently brain levels of inflammatory markers, oxidative parameters, amyloid beta (Aβ), IKKB and NF-kB levels were estimated. Avanafil produced good IKKB and TNFR1 binding ability. It interacted with crucial inhibitory amino acids of IKKB and TNFR1. MD analysis predicted good stability of avanafil with TNFR1 and IKKB. Avanafil 6 mg/kg could significantly improve performance in MWM, PAT and oxidative parameters and reduce Aβ levels and inflammatory markers. As compared to avanafil 3 mg/kg, 6 mg/kg dose was found to exert better efficacy against elevated Aβ , neuroinflammatory cytokines and oxidative markers while improving behavioural parameters. Communicated by Ramaswamy H. Sarma

Differential Contribution of NF-κB Signaling Pathways to CD4+ Memory T Cell Induced Activation of Endothelial Cells.

Endothelial cells (ECs) are important contributors to inflammation in immune-mediated inflammatory diseases (IMIDs). In this study, we examined whether CD4+ memory T (Tm) cells can drive EC inflammatory responses. Human Tm cells produced ligands that induced inflammatory responses in human umbilical vein EC as exemplified by increased expression of inflammatory mediators including chemokines and adhesion molecules. NF-κB, a key regulator of EC activation, was induced by Tm cell ligands. We dissected the relative contribution of canonical and non-canonical NF-κB signaling to Tm induced EC responses using pharmacological small molecule inhibitors of IKKβ (iIKKβ) or NF-κB inducing kinase (iNIK). RNA sequencing revealed substantial overlap in IKKβ and NIK regulated genes (n=549) that were involved in inflammatory and immune responses, including cytokines (IL-1β, IL-6, GM-CSF) and chemokines (CXCL5, CXCL1). NIK regulated genes were more restricted, as 332 genes were uniquely affected by iNIK versus 749 genes by iIKKβ, the latter including genes involved in metabolism, proliferation and leukocyte adhesion (VCAM-1, ICAM-1). The functional importance of NIK and IKKβ in EC activation was confirmed by transendothelial migration assays with neutrophils, demonstrating stronger inhibitory effects of iIKKβ compared to iNIK. Importantly, iIKKβ – and to some extent iNIK - potentiated the effects of currently employed therapies for IMIDs, like JAK inhibitors and anti-IL-17 antibodies, on EC inflammatory responses. These data demonstrate that inhibition of NF-κB signaling results in modulation of Tm cell-induced EC responses and highlight the potential of small molecule NF-κB inhibitors as a novel treatment strategy to target EC inflammatory responses in IMIDs.

Identify promising IKK-β inhibitors: A docking-based 3D-QSAR study combining molecular design and molecular dynamics simulation

Inhibitor of nuclear factor kappa B kinase subunit beta (IKK-β), a specific regulator of nuclear factor-κB (NF-κB), is considered a valid target to design novel drugs to treat rheumatoid arthritis, glomerulonephritis and various cancers. In this study, in order to design and then identify promising compounds targeting IKK-β, a series of reported IKK-β inhibitors were used to develop 3D-QSAR models. Docking-based and minimization-based poses were generated for model construction. CoMSIA model #8 based on docking poses was selected due to its satisfactory internal and external validation results and the sufficient information delivery capability. After a contour map analysis, 41 new designs were depicted based on a graphical design scheme and 25 of them were assessed as eligible for screening. Compound 21MX007 has aroused our attention for its both competitive QSAR-prediction and docking-scoring result. Detailed docking interactions of 21MX007-protein complex were investigated via a deep analysis of docking results and a comparative molecular dynamics simulation. Strong interactions and an extra hydrogen bond which echoes the H-bond requirements of substituent acquired from the design scheme were observed. From MD analysis, 21MX007-protein system was tested. The system was proved to have good stability in terms of a downward trend of RMSD and Rg values and a continuous and stable H-bond interaction and a lower average binding free energy. Thus, compound 21MX007 was successfully identified as a promising IKK-β inhibitor.

Small-molecule IKKβ activation modulator (IKAM) targets MAP3K1 and inhibits pancreatic tumor growth

Activation of inhibitor of nuclear factor NF-κB kinase subunit-β (IKKβ), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKβ knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKβ and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKβ is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKβ in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKβ inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKβ but inhibited the IKKβ kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKβ phosphorylation was inhibited by 39-100, thus we termed it IKKβ activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKβ levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKβ activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

Targeting CaMKII-δ9 Ameliorates Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Inflammation.

CaMKII (Ca2+/calmodulin-dependent kinase II) plays a central role in cardiac ischemia/reperfusion (I/R) injury-an important therapeutic target for ischemic heart disease. In the heart, CaMKII-δ is the predominant isoform and further alternatively spliced into 11 variants. In humans, CaMKII-δ9 and CaMKII-δ3, the major cardiac splice variants, inversely regulate cardiomyocyte viability with the former pro-death and the latter pro-survival. However, it is unknown whether specific inhibition of the detrimental CaMKII-δ9 prevents cardiac I/R injury and, if so, what is the underlying mechanism. Here, we aim to investigate the cardioprotective effect of specific CaMKII-δ9 inhibition against myocardial I/R damage and determine the underlying mechanisms. The role and mechanism of CaMKII-δ9 in cardiac I/R injury were investigated in mice in vivo, neonatal rat ventricular myocytes, and human embryonic stem cell-derived cardiomyocytes. We demonstrate that CaMKII-δ9 inhibition with knockdown or knockout of its feature exon, exon 16, protects the heart against I/R-elicited injury and subsequent heart failure. I/R-induced cardiac inflammation was also ameliorated by CaMKII-δ9 inhibition, and compared with the previously well-studied CaMKII-δ2, CaMKII-δ9 overexpression caused more profound cardiac inflammation. Mechanistically, in addition to IKKβ (inhibitor of NF-κB [nuclear factor-κB] kinase subunit β), CaMKII-δ9, but not δ2, directly interacted with IκBα (NF-κB inhibitor α) with its feature exon 13-16-17 combination and increased IκBα phosphorylation and consequently elicited more pronounced activation of NF-κB signaling and inflammatory response. Furthermore, the essential role of CaMKII-δ9 in myocardial inflammation and damage was confirmed in human cardiomyocytes. We not only identified CaMKII-δ9-IKK/IκB-NF-κB signaling as a new regulator of human cardiomyocyte inflammation but also demonstrated that specifically targeting CaMKII-δ9, the most abundant CaMKII-δ splice variant in human heart, markedly suppresses I/R-induced cardiac NF-κB activation, inflammation, and injury and subsequently ameliorates myocardial remodeling and heart failure, providing a novel therapeutic strategy for various ischemic heart diseases.

IKKα plays a major role in canonical NF-κB signalling in colorectal cells.

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR–Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells, IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

IKKβ Alleviates Neuron Injury in Alzheimer's Disease via Regulating Autophagy and RIPK1-Mediated Necroptosis.

Alzheimer's disease (AD), featured with memory loss and multiple cognitive impairments, is a devastating neurodegenerative disease that affects millions of people in the world, especially the elder people. IKKβ plays important role in the development of neurodegenerative diseases. However, the molecular mechanism of IKKβ, especially related with autophagy and necroptosis, in AD, is still unclear. Here, we studied the function of IKKβ in regulating autophagy and RIPK1-induced necroptosis in SH-SY5Y cells and APP/PS1 mice. By silencing IKKβ in the SH-SY5Y cells, we found that inhibition of IKKβ could promote the RIPK1-induced necroptosis caused by Aβ accumulation as well as suppress the autophagy of SH-SY5Y cells. Furthermore, we discovered that autophagy was significantly enhanced, and RIPK1-induced necroptosis was inhibited when IKKβ was constitutively activated in SH-SY5Y cells. Then, using APP/PS1 mouse model, we demonstrated that silencing IKKβ could significantly enhance the accumulation of Aβ but have not impact on the mice behavior and cognitive ability. Even the controversial results about the role of IKKβ in AD is not fully understood, our results might provide an important potential therapeutic target for slowing AD. .