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

Background: IκB kinase ß (IKKß) 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 IKKß 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 IKKß 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.

This research paper focuses on the development and validation of IκB kinase ß (IKKß) inhibitors. IKKß is a crucial enzyme that plays an important role in the NF-κB signaling pathway, which is involved in many diseases such as inflammatory diseases and cancers. The researchers used computer-aided drug design strategies to identify potential IKKß inhibitors. First, they used a model to screen a large database of chemical compounds. Then, they conducted further tests to pinpoint the ones that could effectively inhibit IKKß. Out of all the tested compounds, one referred to as 'Hit 4' showed the highest inhibitory activity. It was even able to significantly reduce joint inflammation and damage in rat models. Although many drugs targeting IKKß have been developed, none are commercially available yet due to issues with efficacy or safety. Therefore, the findings of this study are significant and could lead to the development of new effective therapeutic agents for inflammatory diseases.

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

The kinase activity of inhibitory κB kinase ß (IKKß) 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 IKKß activity on acetaminophen (APAP)-induced hepatotoxicity remains to be defined. Here, a derivative of caffeic acid benzylamide (CABA) inhibited the kinase activity of IKKß, as did IMD-0354 and sulfasalazine which show therapeutic efficacy against inflammatory diseases through a common mechanism: inhibiting IKKß activity. To understand the importance of IKKß 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 IKKß 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, IKKß 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 IKKß, 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 IKKß activity in sterile inflammation could mitigate APAP-induced hepatotoxicity in mice, and have the potential to complement NAC treatment in APAP overdoses.

Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein.

The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic ß loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

A small molecule UPR modulator for diabetes identified by high throughput screening.

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic ß cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving ß cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.

Suppression of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma through IKKß inhibition.

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKß in regulation of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We showed that cisplatin-resistant HNSCC cells have a stronger ability to migrate and invade, as well as display higher IKKß/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKß, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKß inhibitor, CmpdA, also inhibited cell migration and invasion. Previous studies have already shown that N-Cadherin, an epithelial-mesenchymal transition (EMT) marker, and IL-6, a pro-inflammatory cytokine, play important roles in regulation of HNSCC migration, invasion, and metastasis. We found that cisplatin-resistant HNSCC expressed higher levels of N-Cadherin and IL-6, which were significantly inhibited by CmpdA. More importantly, we showed that CmpdA treatment dramatically abated cisplatin-resistant HNSCC cell metastasis to lungs in a mouse model. Our data demonstrated the crucial role of IKKß in control of migration, invasion, and metastasis, and implicated that targeting IKKß may be a potential therapy for cisplatin-resistant metastatic HNSCC.

Therapeutic potential of IKK-ß inhibitors from natural phenolics for inflammation in cardiovascular diseases.

Cardiovascular disease (CVDs) is a chronic disease with the highest morbidity and mortality in the world. Previous studies have suggested that preventing inflammation serves an efficient role in protection against cardiovascular diseases. Modulation of IKK-ß activity can be used to treat and control CVDs associated with chronic inflammation, which targets the phosphorylation of IκB following the release of the RelA complex, and then translocates to the nucleus, eventually triggering the transcription of several genes that induce chemokines, cytokines, and adhesion molecules. Most importantly, the IκB kinase (IKK) complex is involved in transcriptional activation by phosphorylating the inhibitory molecule IkBα, enabling activation of NF-κB. Phenolic compounds possess cardioprotective potential that may be related to modulating inflammatory responses involved in CVDs. The SystemsDock analysis was used to explore whether 38 active compounds inhibit IKK-ß activity based on literature. Docking results showed that the top docking score of three chemical compounds were icariin, salvianolic acid B, and plantainoside D in all compounds. Icariin, salvianolic acid B, and plantainoside D are the most promising IKKß inhibitors. These phytochemicals could be helpful to find the lead compounds on designing and developing novel cardioprotective agents.

Phytochemicals as potential IKK-ß inhibitor for the treatment of cardiovascular diseases in plant preservation: terpenoids, alkaloids, and quinones.

Modulation of inhibitor kappa B kinase-beta (IKK-ß) kinase activity could be useful for preventing inflammation that serves an efficient role in protection against cardiovascular diseases (CVDs). IKK-ß induces inflammation by activating transcription factor NF-kappa B (NF-κB) through phosphorylation of IκB. Therefore, IKK-ß is considered an interesting target for protecting and treating CVDs. The cardioprotective potential of terpenoids, alkaloids and quinines may be related to modulating inflammatory responses. In this study, the interactions between different classes of inhibitors and IKK-ß were investigated, through the application of SystemsDock. Molecular docking results showed that Diosgenin and Ginsenoside Re were the top docking score compounds. Diosgenin and Ginsenoside Re are the most promising IKK-ß inhibitors in terpenoids, alkaloids, and quinones. Diosgenin and Ginsenoside Re could be helpful to find the lead compounds on designing and developing novel cardioprotective agents.

Abietic acid suppresses non-small-cell lung cancer cell growth via blocking IKKß/NF-κB signaling.

Background: Abietic acid (AA) is one of the terpenoids, which are multifunctional natural compounds. It has been reported that AA possesses favorable therapeutic effects on inflammation and obesity. Method: In the present study, we determined the inhibitory effect of AA on the proliferation and growth of non-small-cell lung cancer (NSCLC) cell lines for the first time. Then, flow cytometry and Western blot analysis were applied to determine the cell apoptosis and cell cycle. Finally, surface plasmon resonance, molecular docking and molecular dynamics (MD) simulation were performed to explore the underlying molecular mechanisms. Results: In vitro experiments indicated that AA displays significant anti-proliferative, cell cycle arresting and pro-apoptotic activities. Mechanistically, AA abrogated tumor necrosis factor-α induced phosphorylation of IκB kinase (IKKα/ß) (Ser176/180) and IkBα (Ser32), and inhibited the nuclear translocation of nuclear factor-κB. Moreover, we found that the activities of AA against NSCLC cells were mediated by its IKKß inhibition. Molecular docking and MD simulations demonstrated that the mechanism of action between AA and IKKß was through hydrophobic interactions. Conclusion: Our data indicate that AA could be a promising lead compound for the discovery of novel IKKß inhibitors and potential agents for the treatment of NSCLC.

Synthesis and anti-tumor activity of EF24 analogues as IKKß inhibitors.

EF24 is an IKKß inhibitor (IC50: 72 µM) containing various anti-tumor activities. In this study, a series of EF24 analogs targeting IKKß were designed and synthesized. Several IKKß inhibitors with better activities than EF24 were screened out and B3 showed best IKKß inhibitory (IC50: 6.6 µM). Molecular docking and dynamic simulation experiments further confirmed this inhibitory effect. B3 obviously suppressed the viability of Hela229, A549, SGC-7901 and MGC-803 cells. Then, in SGC-7901 and MGC-803 cells, B3 blocked the NF-κB signal pathway by inhibiting IKKß phosphorylation, and followed arrested the cell cycle at G2/M phase by suppressing the Cyclin B1 and Cdc2 p34 expression, induced the cell apoptosis by down-regulating Bcl-2 protein and up-regulating cleaved-caspase3. Moreover, B3 significantly reduced tumor growth and suppressed the IKKß-NF-κB signal pathway in SGC-7901 xenograft model. In total, this study present a potential IKKß inhibitor as anti-tumor precursor.

Astrocyte IKKß/NF-κB signaling is required for diet-induced obesity and hypothalamic inflammation.

OBJECTIVE: Obesity and high fat diet (HFD) consumption in rodents is associated with hypothalamic inflammation and reactive gliosis. While neuronal inflammation promotes HFD-induced metabolic dysfunction, the role of astrocyte activation in susceptibility to hypothalamic inflammation and diet-induced obesity (DIO) remains uncertain. METHODS: Metabolic phenotyping, immunohistochemical analyses, and biochemical analyses were performed on HFD-fed mice with a tamoxifen-inducible astrocyte-specific knockout of IKKß (GfapCreERIkbkbfl/fl, IKKß-AKO), an essential cofactor of NF-κB-mediated inflammation. RESULTS: IKKß-AKO mice with tamoxifen-induced IKKß deletion prior to HFD exposure showed equivalent HFD-induced weight gain and glucose intolerance as Ikbkbfl/fl littermate controls. In GfapCreERTdTomato marker mice treated using the same protocol, minimal Cre-mediated recombination was observed in the mediobasal hypothalamus (MBH). By contrast, mice pretreated with 6 weeks of HFD exposure prior to tamoxifen administration showed substantially increased recombination throughout the MBH. Remarkably, this treatment approach protected IKKß-AKO mice from further weight gain through an immediate reduction of food intake and increase of energy expenditure. Astrocyte IKKß deletion after HFD exposure-but not before-also reduced glucose intolerance and insulin resistance, likely as a consequence of lower adiposity. Finally, both hypothalamic inflammation and astrocytosis were reduced in HFD-fed IKKß-AKO mice. CONCLUSIONS: These data support a requirement for astrocytic inflammatory signaling in HFD-induced hyperphagia and DIO susceptibility that may provide a novel target for obesity therapeutics.

Identification and characterization of potent, selective and metabolically stable IKKß inhibitor.

We have previously reported the identification of a rhodanine compound (1) with well-balanced inhibitory activity against IKKß and collagen-induced TNFα activated cells. However, we need more optimized compounds because of its instability over plasma and microsome. As part of a program directed toward the optimization of IKKß inhibitor, we modified a substituent of parent compound to a series of functional groups. Among substituted compounds, fluorine substituent (12) on the para position of phenyl ring restored the stability toward plasma and microsome while retaining inhibitory potency and selectivity against IKKß over other kinases. Also, we have demonstrated that compound 12 is an ATP non-competitive inhibitor and safe enough to apply to animal experiment from an acute toxicity test.

A quinoxaline urea analog uncouples inflammatory and pro-survival functions of IKKß.

Activation of the NF-κB pathway is causally linked to initiation and progression of diverse cancers. Therefore, IKKß, the key regulatory kinase of the canonical NF-κB pathway, should be a logical target for cancer treatment. However, existing IKKß inhibitors are known to induce paradoxical immune activation, which limits their clinical usefulness. Recently, we identified a quinoxaline urea analog 13-197 as a novel IKKß inhibitor that delays tumor growth without significant adverse effects in xenograft tumor models. In the present study, we found that 13-197 had little effect on LPS-induced NF-κB target gene induction by primary mouse macrophages while maintaining considerable anti-proliferative activities. These characteristics may explain absence of inflammatory side effects in animals treated with 13-197. Our data also demonstrate that the inflammation and proliferation-related functions of IKKß can be uncoupled, and highlight the utility of 13-197 to dissect these downstream pathways.

Mutation of cysteine 46 in IKK-beta increases inflammatory responses.

Activation of IκB kinase ß (IKK-ß) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-ß-NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-ß. However, mutations in IKK-ß have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-ß inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-ß kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-ß C46A transgenic mouse model. We show that a novel IKK-ß inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-ß C46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-ß.

Non-alcoholic Fatty Liver Disease and Metabolic Syndrome-Position Paper of the Indian National Association for the Study of the Liver, Endocrine Society of India, Indian College of Cardiology and Indian Society of Gastroenterology.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with metabolic syndrome. Prevalence of metabolic risk factors including diabetes mellitus, obesity, etc. is rapidly increasing in India putting this population at risk for NAFLD. Patients with NAFLD are at increased risk for liver-related morbidity and mortality and also cardiovascular disease risk and increased incidence of diabetes mellitus on long-term follow-up. Management of patients with NAFLD may require a multi-disciplinary approach involving not only the hepatologists but also the internists, cardiologists, and endocrinologists. This position paper which is a combined effort of the Indian National Association for Study of the Liver (INASL), Endocrine Society of India (ESI), Indian College of Cardiology (ICC) and the Indian Society of Gastroenterology (ISG) defines the spectrum of NAFLD and the association of NAFLD with insulin resistance and metabolic syndrome besides suggesting preferred approaches for the diagnosis and management of patients with NAFLD in the Indian context.

Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK, p38, and nuclear factor-κB signaling pathways.

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 µg/ml), tigecycline (50 µg/ml), or doxycycline (50 µg/ml) were added after treatment with LPS (10 µg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1ß was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.

Retrospective TREC testing of newborns with Severe Combined Immunodeficiency and other primary immunodeficiency diseases.

In Manitoba, Canada, the overall incidence of Severe Combined Immunodeficiency (SCID) is three-fold higher than the national average, with SCID overrepresented in two population groups: Mennonites and First Nations of Northern Cree ancestries. T-cell receptor excision circle (TREC) assay is being used increasingly for neonatal screening for SCID in North America. However, the majority of SCID patients in Manitoba are T-cell-positive. Therefore it is likely that the TREC assay will not identify these infants. The goal of this study was to blindly and retrospectively perform TREC analysis in confirmed SCID patients using archived Guthrie cards. Thirteen SCID patients were tested: 5 T-negative SCID (3 with adenosine deaminase deficiency, 1 with CD3δ deficiency, and 1 unclassified) and 8 T-positive SCID (5 with zeta chain-associated protein kinase (ZAP70) deficiency and 3 with inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKKß) deficiency). As a non-SCID patient group, 5 Primary Immunodeficiency Disease (PID) patients were studied: 1 T-negative PID (cartilage-hair hypoplasia) and 4 T-positive PID (2 common immune deficiency (CID), 1 Wiskott-Aldrich syndrome, and 1 X-linked lymphoproliferative disease). Both patient groups required hematopoietic stem cell transplantation. In addition, randomly-selected de-identified controls (n = 982) were tested. Results: all T-negative SCID and PID had zero TRECs. Low-TRECs were identified in 2 ZAP70 siblings, 1 CID patient as well as 5 preterm, 1 twin, and 4 de-identified controls. Conclusions: TREC method will identify T-negative SCID and T-negative PID. To identify other SCID babies, newborn screening in Manitoba must include supplemental targeted screening for ethnic-specific mutations.