The non-steroidal anti-inflammatory drug indomethacin activates the eIF2α kinase PKR, causing a translational block in human colorectal cancer cells

The NSAID (non-steroidal anti-inflammatory drug) indomethacin, a cyclo-oxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against CRC (colorectal cancer) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. In the present study we show that indomethacin selectively activates the dsRNA (double-stranded RNA)-dependent protein kinase PKR in a cyclo-oxygenase-independent manner, causing rapid phosphorylation of eIF2α (the α-subunit of eukaryotic translation initiation factor 2) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of the eIF2α kinases PERK (PKR-like endoplasmic reticulum-resident kinase), GCN2 (general control non-derepressible-2) and HRI (haem-regulated inhibitor kinase), and induced eIF2α phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in vitro, an effect reversed by the PKR inhibitor 2-aminopurine, suggests a direct effect of the drug in kinase activation. The results of the present study identify PKR as a novel target of indomethacin, suggesting new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.     

Equithesin: A hibernation-inducing drug?

1. Hibernating insectivore species (hedgehogs) and non-hibernating rodents (guinea pig and rat) were anaesthetized with ‘equithesin’ (a mixture of chloral hydrate, magnesium sulphate, and pentobarbitone sodium).2. The physiological responses shown by the hedgehogs were similar to those observed in hedgehogs during a natural or cold-induced hibernation.3. These responses included a strong reduction in body temperature, heart rate, respiration and oxygen consumption, and brain activity.4. Such responses to equithesin were not observed in the non-hibemating rodent species.5. These results suggest that equithesin is a potential tool for hibernation research.     

Chemogenomics: drug discovery's panacea?

Chemogenomics aims towards the systematic identification of small molecules that interact with the products of the genome and modulate their biological function. This Opinion article summarizes the different knowledge-based chemogenomics strategies that are followed and outlines the challenges and opportunities that will impact drug discovery. Chemogenomics aims towards the systematic identification of small molecules that interact with the products of the genome and modulate their biological function. While historically the approach is based on efforts that systematically explore target gene families like kinases, today additional knowledge-based systematization principles are followed within early drug discovery projects which aim to biologically validate the targets and to identify starting points for chemical lead optimization. While the expectations of chemogenomics are very high, the reality of drug discovery is quite sobering with very high project attrition rates. This article summarizes the different knowledge-based chemogenomics strategies that are followed and outlines the challenges and potential opportunities that will impact drug discovery.     

Ashwagandha—An ancient Indian drug

Ashwagandha, the root ofWithania somnifera Dunal, is an ancient drug used in India by practitioners of Ayurvedic mediicne and as a household remedy. The drug is now official in the Indian Pharmacopoeia. Results of a survey regarding the cultivation, collection and marketing of this drug are described.     

The role of “anti-inflammatory” cytokines in axon regeneration

The injured central and peripheral nervous system (CNS and PNS) are difficult to regenerate due to the presence of growth inhibitory molecules which are upregulated around the lesion site. In addition, a strong inflammatory response triggering the production of so-called “pro”- and “anti-inflammatory” cytokines, adds to this dilemma. Both pro- and anti-inflammatory cytokines are involved in the regulation of diverse signaling pathways. One of the main aims to induce regeneration is to promote axonal outgrowth and stimulate the formation of new connections. Anti-inflammatory cytokines as modulators of neurite plasticity and outgrowth are of pivotal importance in neuroregeneration with different effects reported. Here we summarize the most relevant information about IL-4, IL-10, IL-13, LIF and TGF-β focusing on their direct and indirect role in axonal outgrowth.     

Molecular mechanism of an adverse drug–drug interaction of allopurinol and furosemide in gout treatment

Gout patients receiving a combination of allopurinol and furosemide require higher allopurinol doses to achieve the target serum urate (SU) of <6 mg/dl (Stamp et al., 2012) [1]. Our study aimed to identify the molecular basis for this observation. We used a fluorimetric assay to determine the impact of furosemide and oxypurinol (the active metabolite of allopurinol) on xanthine oxidase (XO) activity. Immunoblot analysis quantified expression of XO and AMP-kinase (AMPK) in drug-treated human liver (HepG2) and primary kidney (HRCE) cells. In silico analysis identified miR-448 as a potential XO-regulator, whose expression level in HepG2 cells was examined by qPCR.     

Analysis and prediction of drug–drug interaction by minimum redundancy maximum relevance and incremental feature selection

Drug–drug interaction (DDI) defines a situation in which one drug affects the activity of another when both are administered together. DDI is a common cause of adverse drug reactions and sometimes also leads to improved therapeutic effects. Therefore, it is of great interest to discover novel DDIs according to their molecular properties and mechanisms in a robust and rigorous way. This paper attempts to predict effective DDIs using the following properties: (1) chemical interaction between drugs; (2) protein interactions between the targets of drugs; and (3) target enrichment of KEGG pathways. The data consisted of 7323 pairs of DDIs collected from the DrugBank and 36,615 pairs of drugs constructed by randomly combining two drugs. Each drug pair was represented by 465 features derived from the aforementioned three categories of properties. The random forest algorithm was adopted to train the prediction model. Some feature selection techniques, including minimum redundancy maximum relevance and incremental feature selection, were used to extract key features as the optimal input for the prediction model. The extracted key features may help to gain insights into the mechanisms of DDIs and provide some guidelines for the relevant clinical medication developments, and the prediction model can give new clues for identification of novel DDIs.     

From exotic spice to modern drug?

The global demand for more affordable therapeutics and concerns about side effects of commonly used drugs are refocusing interest on Eastern traditional medicines, particularly those of India and China.     

The ‘president’s drug’

Netherlands Heart Journal -     

Will genomics revolutionize antimicrobial drug discovery?

Utilizing genome sequence data from bacterial and fungal pathogens for the discovery of new antimicrobial agents has received considerable attention, both practical and critical, from the pharmaceutical and biotechnological communities. Although no new drugs derived from genomics-based discovery have been reported to be in a development pipeline, the utilization of genomics has revolutionized many aspects of drug discovery. The application, utility, opportunity, and challenges afforded by many of these new approaches are discussed.     

Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24 h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.     

Repositioning drugs for inflammatory disease – fishing for new anti-inflammatory agents

Inflammation is an important and appropriate host response to infection or injury. However, dysregulation of this response, with resulting persistent or inappropriate inflammation, underlies a broad range of pathological processes, from inflammatory dermatoses to type 2 diabetes and cancer. As such, identifying new drugs to suppress inflammation is an area of intense interest. Despite notable successes, there still exists an unmet need for new effective therapeutic approaches to treat inflammation. Traditional drug discovery, including structure-based drug design, have largely fallen short of satisfying this unmet need. With faster development times and reduced safety and pharmacokinetic uncertainty, drug repositioning – the process of finding new uses for existing drugs – is emerging as an alternative strategy to traditional drug design that promises an improved risk-reward trade-off. Using a zebrafish in vivo neutrophil migration assay, we undertook a drug repositioning screen to identify unknown anti-inflammatory activities for known drugs. By interrogating a library of 1280 approved drugs for their ability to suppress the recruitment of neutrophils to tail fin injury, we identified a number of drugs with significant anti-inflammatory activity that have not previously been characterized as general anti-inflammatories. Importantly, we reveal that the ten most potent repositioned drugs from our zebrafish screen displayed conserved anti-inflammatory activity in a mouse model of skin inflammation (atopic dermatitis). This study provides compelling evidence that exploiting the zebrafish as an in vivo drug repositioning platform holds promise as a strategy to reveal new anti-inflammatory activities for existing drugs.KEY WORDS: Drug repositioning, Zebrafish, Inflammation, Neutrophil, Atopic dermatitis, Immunity     

Does conjugation of antioxidants improve their antioxidative/anti-inflammatory potential?

A series of symmetric and asymmetric spermine (SPM) conjugates with all-trans-retinoic acid (ATRA), acitretin (ACI), (E)-3-(trioxsalen-4′-yl)acrylic acid (TRAA) and l-DOPA, amides of ACI, l-DOPA and TRAA with 1-aminobutane, benzylamine, dopamine and 1,12-diaminobutane as well as hybrid conjugates of O,O′-dimethylcaffeic acid (DMCA) with TRAA or N-fumaroyl-indole-3-carboxanilide (FICA) and 2-(2-aminoethoxy)ethanol were synthesized and their antioxidant properties were studied. The reducing activity (RA)% of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay and found to be in the range 0–92(20 min)%/96(60 min)% at 100 μM, the most powerful being the conjugates l-DOPA-SPM-l-DOPA (8, RA = 89%/96%) and l-DOPA-dopamine (13, RA = 92%/92%). Conjugate DMCA-NH(CH₂CH₂O)₂-FICA (14) was the most powerful LOX inhibitor with IC₅₀ 33.5 μM, followed by the conjugates ACI-NHCH₂Ph (10, IC₅₀ 40.5 μM), ACI-SPM-TRAA (7, IC₅₀ 41.5 μM), DMCA-NH(CH₂CH₂O)₂-TRAA (15, IC₅₀ 65 μM), 13 (IC₅₀ 81.5 μM) and ACI-dopamine (11, IC₅₀ 87 μM). The most potent inhibitors of lipid peroxidation at 100 μM were the conjugates 15 (98%) and ACI-SPM-ACI (4, 97%) whereas all other compounds showed activities comparable or lower than trolox. The most interesting compounds, namely ATRA-SPM-ATRA (3), 4, 10, 11 and 15, as well as unconjugated compounds such as ATRA and dopamine, were studied for their anti-inflammatory activity in vivo on rat paw oedema induced by Carrageenan and found to exhibit, for doses of 0.01 mmol/mL of conjugates per Kg of rat body weight, weaker anti-inflammatory activities (3.6–40%) than indomethacin (47%) with conjugate 3 being the most potent (40%) in this series of compounds. The cytocompatibility of selected compounds was evaluated by the viability of RAMEC cells in the presence of different concentrations (0.5–50 μM) of the compounds. Conjugates 3 (IC₅₀ 2.6 μM) and 4 (IC₅₀ 4.7 μM) were more cytotoxic than the corresponding unconjugated retinoids ATRA (IC₅₀ 18.3 μM) and ACI (IC₅₀ 14.6 μM), whereas conjugate 15 (IC₅₀ 12.9 μM) was less cytotoxic than either DCSP (IC₅₀ 11.3 μM) or the tert-butyl ester of TRAA (IC₅₀ 2.9 μM).     

High salt induces anti-inflammatory MΦ2-like phenotype in peripheral macrophages

Macrophages play a critical role in inflammation and antigen-presentation. Abnormal macrophage function has been attributed in autoimmune diseases and cancer progression. Recent evidence suggests that high salt tissue micro-environment causes changes in macrophage activation. In our current report, we studied the role of extracellular sodium chloride on phenotype changes in peripheral circulating monocyte/macrophages collected from healthy donors. High salt (0.2 M NaCl vs basal 0.1 M NaCl) treatment resulted in a decrease in MΦ1 macrophage phenotype (CD11b⁺CD14^highCD16^low) from 77.4±6.2% (0.1 M) to 29.3±5.7% (0.2 M, p<0.05), while there was an increase in MΦ2 macrophage phenotype (CD11b⁺ CD14^lowCD16^high) from 17.2±5.9% (0.1 M) to 67.4±9.4% (0.2 M, p<0.05). ELISA-based cytokine analysis demonstrated that high salt treatment induced decreased expression of in the MΦ1 phenotype specific pro-inflammatory cytokine, TNFα (3.3 fold), IL-12 (2.3 fold), CCL-10 (2 fold) and CCL-5 (3.8 fold), but conversely induced an enhanced expression MΦ2-like phenotype specific anti-inflammatory cytokine, IL-10, TGFβ, CCL-17 (3.7 fold) and CCR-2 (4.3 fold). Further high salt treatment significantly decreased phagocytic efficiency of macrophages and inducible nitric oxide synthetase expression. Taken together, these data suggest that high salt extracellular environment induces an anti-inflammatory MΦ2-like macrophage phenotype with poor phagocytic and potentially reduced antigen presentation capacity commonly found in tumor microenvironment.