Conversion Of Arachidonic Acid Research Articles

Fenamates and ibuprofen as foundational components in the synthesis of innovative, targeted COX-2 anti-inflammatory drugs, undergoing thorough biopharmacological assessments and in-silico computational studies

Cyclooxygenase-2 plays a vital role in inflammation by catalyzing arachidonic acid conversion toward prostaglandins, making it a prime therapeutic objective. Selective COX-2 inhibitors represent significant progress in anti-inflammatory therapy, offering improved efficacy and fewer side effects. This study describes the synthesis of novel anti-inflammatory compounds from established pharmaceutically marketed agents like fenamates III-V and ibuprofen VI. Through rigorous in vitro testing, compounds 7b-c, and 12a-b demonstrated substantial in vitro selective inhibition, with IC50 values of 0.07 to 0.09 μM, indicating potent pharmacological activity. In vivo assessment, particularly focusing on compound 7c, revealed significant anti-inflammatory effects. Markedly, it demonstrated the highest inhibition of paw thickness (58.62 %) at the 5-hr mark compared to the carrageenan group, indicating its potency in mitigating inflammation. Furthermore, it exhibited a rapid onset of action, with a 54.88 % inhibition observed at the 1-hr mark. Subsequent comprehensive evaluations encompassing analgesic efficacy, histological characteristics, and toxicological properties indicated that compound 7c did not induce gastric ulcers, in contrast to the ulcerogenic tendency associated with mefenamic acid. Moreover, compound 7c underwent additional investigations through in silico methodologies such as molecular modelling, field alignment, and density functional theory. These analyses underscored the therapeutic potential and safety profile of this novel compound, warranting further exploration and development in the realm of pharmaceutical research.

Design, Synthesis, In vitro and In vivo Evaluation of New Imidazo[1,2-a]pyridine Derivatives as Cyclooxygenase-2 Inhibitors.

Cyclooxygenase-2 (COX-2), the key enzyme in the arachidonic acid conversion to prostaglandins, is one of the enzymes associated with different pathophysiological conditions, such as inflammation, cancers, Alzheimer's, and Parkinson's disease. Therefore, COX-2 inhibitors have emerged as potential therapeutic agents in these diseases. The objective of this study was to design and synthesize novel imidazo[1,2-a]pyridine derivatives utilizing rational design methods with the specific aim of developing new potent COX-2 inhibitors. Additionally, we sought to investigate the biological activities of these compounds, focusing on their COX-2 inhibitory effects, analgesic activity, and antiplatelet potential. We aimed to contribute to the development of selective COX-2 inhibitors with enhanced therapeutic benefits. Docking investigations were carried out using AutoDock Vina software to analyze the interaction of designed compounds. A total of 15 synthesized derivatives were obtained through a series of five reaction steps. The COX-2 inhibitory activities were assessed using the fluorescent Cayman kit, while analgesic effects were determined through writing tests, and Born's method was employed to evaluate antiplatelet activities. The findings indicated that the majority of the tested compounds exhibited significant and specific inhibitory effects on COX-2, with a selectivity index ranging from 51.3 to 897.1 and IC50 values of 0.13 to 0.05 μM. Among the studied compounds, derivatives 5e, 5f, and 5j demonstrated the highest potency with IC50 value of 0.05 μM, while compound 5i exhibited the highest selectivity with a selectivity index of 897.19. In vivo analgesic activity of the most potent COX-2 inhibitors revealed that 3-(4-chlorophenoxy)-2-[4-(methylsulfonyl) phenyl] imidazo[1,2-a]pyridine (5j) possessed the most notable analgesic activity with ED50 value of 12.38 mg/kg. Moreover, evaluating the antiplatelet activity showed compound 5a as the most potent for inhibiting arachidonic acidinduced platelet aggregation. In molecular modeling studies, methylsulfonyl pharmacophore was found to be inserted in the secondary pocket of the COX-2 active site, where it formed hydrogen bonds with Arg-513 and His-90. The majority of the compounds examined demonstrated selectivity and potency as inhibitors of COX-2. Furthermore, the analgesic effects observed of potent compounds can be attributed to the inhibition of the cyclooxygenase enzyme.

Abstract 1711: Rearranged active enhancers and promoter-enhancer contacts promote lipid metabolic reprogramming during secondary trastuzumab adaptation of HER2 positive breast cancer

Abstract Background: The development of secondary trastuzumab resistance signifies an evolutionary adaptation within HER2-positive breast cancer during anti-HER2 treatment. Our research reveals a comprehensive metabolic reprogramming, with a particular emphasis on the synthesis of unsaturated fatty acids and the degradation of arachidonic acid, accompanying this adaptation. Non-mutational epigenetic variations, particularly alterations in active enhancers, coupled with the rearrangement of enhancer-promoter contacts, demonstrate robust correlations with lipid metabolic reprogramming. These findings suggest that non-mutational epigenetic variations play a crucial role in driving the adaptation to trastuzumab. Materials and Methods: In a prior investigation, we successfully generated secondary trastuzumab-resistant SKBR3_HR cells. In our current study, total RNA was extracted from both trastuzumab-resistant SKBR3_HR and trastuzumab-sensitive SKBR3 cells for transcriptome analysis. Additionally, mass spectrometry was employed to analyze cellular metabolism in both resistant and sensitive cell populations. Simultaneously, we utilized the CUT&Tag kit with an anti-H3K27ac antibody to create a sequencing library for active enhancer measurement. Furthermore, the Micro-C kit was applied to generate a sequencing library for genome contact measurement. Results: In our study, secondary trastuzumab-adaptive SKBR3_HR cells demonstrated a reduction in cellular unsaturated fatty acids, with more than 110 UFAs or UFA-containing lipids showing decreases, while only 36 exhibited increases. This phenomenon may be attributed to the downregulation of SCD, FADS2, and HACD2. Simultaneously, the upregulation of PTGS1 and PTGES may contribute to a higher conversion of arachidonic acid to prostaglandin E2 (PGE2), resulting in immune inhibition. In SKBR3 and SKBR3_HR cells, 10,955 and 10,889 non-promoter H3K27me3 peaks, respectively, were identified as active enhancer regions. Among them, 379 enhancers became silent, and 313 transitioned into an active state during the formation of trastuzumab adaptation. Trastuzumab adaptation led to an increase in intra-chromosomal interactions, particularly short-distance contacts (< 1MB). By employing the activity-by-contact (ABC) algorithm, we observed significant variant contacts between PTGES and SCD promoters and enhancers within 1MB genomic regions in SKBR3_HR cells compared to SKBR3 cells. Conclusion: The formation of secondary trastuzumab adaptation involves the rearrangement of active enhancers and promoter-enhancer contacts, serving as an effective force to drive lipid metabolic reprogramming, especially the synthesis of unsaturated fatty acids and PGE2 production, reshaping the cellular fatty acid composition and anti-tumor immunity. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Rearranged active enhancers and promoter-enhancer contacts promote lipid metabolic reprogramming during secondary trastuzumab adaptation of HER2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1711.

The importance of cyclooxigenase in dentistry

Aim: Cyclooxygenase enzymes (COX) catalyze the conversion of arachidonic acid to prostaglandins and thromboxanes during pain and inflammation conditions. These enzymes have also been linked to several other conditions and diseases, and hence, in dentistry, it is crucial to identify the processes that increase the levels of these mediators. This paper aims to describe the significance of COX in dental practice through a narrative review. Methods: Articles relating to COX upregulation published in English and Spanish over the last 51 years in databases such as EBSCO, Google Scholar, Science Direct, PubMed, and Web of Science; were analyzed. Results: A total of 115 articles demonstrating the relationship between COX upregulation and multiple conditions and diseases of importance in prosthodontics, periodontics, oral pathology, orthodontics, and endodontics were included. Conclusions: COX upregulation is related to inflammatory and malignant diseases in oral tissues, such as periodontitis, pulpitis, and oral cancer, nevertheless, its expression is advantageous in other fields of study such as orthodontics. Additionally, is well documented that dental materials provoke an undesired increase in COX expression, which could be a significant factor that directly affects pulpal health.

Study on the mechanism of Panax notoginseng-Salvia miltiorrhiza herb pair on invigorating blood circulation and eliminating blood stasis by blocking the conversion of arachidonic acid to prostaglandin.

We combined untargeted and targeted metabolomics to explore the mechanism of blood circulation and blood stasis activation in the traditional Chinese herb pair Panax notoginseng-Salvia miltiorrhiza (PS). In this study, the right hind limb of SD rats was struck by a 1kg weight, causing traumatic blood stasis (TBS) model, then the rats were gavaged with PS (at ratios of 1:0, 0:1, 3:1, 1:1, and 1:3) for 5 consecutive days. At the end of treatment, blood samples were collected for blood rheology and metabolomics analysis, and muscle tissues of injured limbs were used for HE staining and q-PCR analysis. The results showed that different ratios of PS reduced swelling and improved stasis and blood viscosity in the injured limbs of rats, and intervened in metabolism by modulating 11, 11, 17, 15, and 13 differential metabolites, respectively. The PS (3:1) shows the best treatment effect and the most differential metabolites regression. Targeted metabolomics shows that PS (3:1) can increase the content of AA, and reduce the content of PGF2-α by down-regulating the expression of enzymes Ptgs1 and Cbrl12 and up-regulating the expression of enzyme Hpgd. These results suggested that the PS herb pair exerts its blood stasis activating effects by blocking the conversion of arachidonic acid to prostaglandins.

Theoretical Study of the Arachidonic Acid Conversion into Leukotriene A4 Catalyzed by Human 5-Lipoxygenase: Hydroperoxidation and Epoxidation Mechanisms and Arachidonic Acid Active Site Access

Theoretical Study of the Arachidonic Acid Conversion into Leukotriene A₄ Catalyzed by Human 5-Lipoxygenase: Hydroperoxidation and Epoxidation Mechanisms and Arachidonic Acid Active Site Access

Nonsteroidal Anti-inflammatory Drug (NSAID) Toxicity in Emergency Room

Nonsteroidal anti-inflammatory medicines, collectively referred to as NSAIDs in academic context, exhibit chemical diversity while concurrently manifesting comparable therapeutic and harmful effects. All pharmaceutical substances under this particular category function by diminishing inflammation, alleviating pain, and reducing fever by means of inhibiting enzymes responsible for the creation of endoperoxides, often referred to as cyclooxygenase (COX) enzymes. The two cyclooxygenase isozymes, COX-1 and COX-2, are responsible for the conversion of arachidonic acid into its endoperoxide metabolites. These metabolites include prostacyclin, prostaglandins, and thromboxane, each exhibiting a wide range of biological activities such as inflammation, regulation of smooth muscle tone, and promotion of thrombosis. The COX-1 enzyme is consistently produced and is recognized as the main provider of prostanoids required for maintaining physiological balance, such as safeguarding the stomach epithelium. In contrast, the COX-2 enzyme is capable of being induced, leading to a substantial increase in the synthesis of prostanoids in circumstances characterized by stress and inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) may induce toxicity via the same pharmacological mechanisms that contribute to their therapeutic efficacy. This review aims to discuss NSAIDs toxicity in details, its symptoms, and possible management in the emergency room.

Long noncoding RNAs and their complex role in shaping and regulating arachidonic acid metabolism: Learning to love the (not-really) junk.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators in numerous biological processes. The arachidonic acid (AA) metabolic pathway is a fundamental biochemical pathway responsible for the enzymatic conversion of AA, a 20-carbon omega-six polyunsaturated fatty acid, into a variety of potent lipid signaling molecules known as eicosanoids. Eicosanoids are produced through the cyclooxygenase and lipoxygenase arms of the AA pathway and have diverse biological roles in both healthy and disease states, including cancer and inflammatory diseases. Cyclooxygenase 2 (COX-2), the inducible, rate-limiting enzyme of the cyclooxygenase arm, produces two main forms of eicosanoids: prostaglandins and thromboxanes. AA metabolized through the lipoxygenase arm by the action of 5-lipoxygenase (ALOX5) produces eicosanoids known as leukotrienes. COX-2 and ALOX5 gene expression are regulated through many different lncRNAs and microRNA (miRNA)-mediated mechanisms. As previously reviewed, noncoding RNAs affect transcription, splicing, alternative polyadenylation, messenger RNA stability, translation, and miRNA regulation of COX-2 and ALOX5 (Lutz and Cornett, 2013, Wiley Interdisciplinary Reviews. RNA, 4(5), 593-605). This current review discusses the intricate roles of lncRNAs, including MALAT1, NEAT1, HOTAIR, PACER, and others, in modulating the AA pathway. In this review update, we will delve into advancements in our understanding of AA gene expression regulation. We will explore the mechanisms of lncRNAs and their associated miRNAs and proteins known to regulate key components of the AA signaling pathway. We will also discuss the therapeutic potential of targeting lncRNA-mediated regulation, with a focus on modulating COX-2 and ALOX5 activity and downstream eicosanoid production for applications in inflammatory and oncological conditions. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease.

Exosomes drive ferroptosis by stimulating iron accumulation to inhibit bacterial infection in crustaceans

Ferroptosis, characterized by iron-dependent cell death, has recently emerged as a critical defense mechanism against microbial infections. The present study aims to investigate the involvement of exosomes in the induction of ferroptosis and the inhibition of bacterial infection in crustaceans. Our findings provide compelling evidence for the pivotal role of exosomes in the immune response of crustaceans, wherein they facilitate intracellular iron accumulation and activate the ferroptotic pathways. Using RNA-seq and bioinformatic analysis, we demonstrate that cytochrome P450 (CYP) can effectively trigger ferroptosis. Moreover, by conducting an analysis of exosome cargo proteins, we have identified the participation of six-transmembrane epithelial antigen of prostate 4 (STEAP4) in the regulation of hemocyte ferroptotic sensitivity. Subsequent functional investigations unveil that STEAP4 enhances cellular Fe2+ levels, thereby triggering Fenton reactions and accelerating CYP-mediated lipid peroxidation, ultimately culminating in ferroptotic cell death. Additionally, the Fe2+-dependent CYP catalyzes the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid (20-HETE), which activates the peroxisome proliferator-activated receptor (PPAR). Consequently, the downstream target of PPAR, cluster of differentiation 36 (CD36), promotes intracellular fatty acid accumulation, lipid peroxidation, and ferroptosis. These significant findings shed light on the immune defense mechanisms employed by crustaceans and provide potential strategies for combating bacterial infections in this species.

Identification of selective 5-LOX and FLAP inhibitors as novel anti-inflammatory agents by ligand-based virtual screening

Inflammation is a multifaceted biological process in which the conversion of arachidonic acid to eicosanoids, including prostaglandins and leukotrienes (LTs), plays a crucial role. 5-Lipoxygenase (5-LOX) is a key enzyme in cellular LT biosynthesis, and it is supported by the accessory protein 5-lipoxygenase-activating protein (FLAP). Pharmacological interventions to modulate LTs aim at either decreasing their biosynthesis or at mitigating their biological effects. Therefore, inhibiting 5-LOX or FLAP represents a useful strategy to reduce inflammation. Herein we present the identification and pharmacological evaluation of novel inhibitors targeting 5-LOX or FLAP. By means of a ligand-based virtual screening approach, we selected 38 compounds for in vitro assays. Among them, ALR-38 exhibits direct 5-LOX inhibition, while ALR-6 and ALR-27 showed potential as FLAP inhibitors. These latter not only reduced LT production but also promoted the generation of specialized pro-resolving mediators in specific human macrophage phenotypes. Interestingly, the identified compounds turned out to be selective for their respective targets, as none of them displayed activity towards microsomal prostaglandin E2 synthase-1 and soluble epoxide hydrolase, which are other proteins involved in eicosanoid biosynthesis. Thus, these compounds are endowed with potential therapeutic utility in mitigating inflammatory responses and might offer a venue for tackling inflammation-based disorders.

In silico molecular docking of cyclooxygenase (COX-2), ADME-toxicity and in vitro evaluation of antioxidant and anti-inflammatory activities of marine macro algae.

The marine ecosystem harbors unique and diverse bioactive compounds that can offer a vast repertoire of molecules with therapeutic properties. In the present study, four different species of red marine seaweeds were analyzed for its phytoconstituents and the potent antioxidant and anti-inflammatory activity of the methanolic extracts were screened and determined. The results revealed that, among the 4 samples, G. corticata, scored a good antioxidant potential by DPPH (67.61 ± 1.23%, IC50 = 577.7µg) and metal chelation assay (29.40 ± 0.32%, IC50 = 1684µg). The anti-inflammatory analysis has shown that, H. dialata was found to exhibit maximum inhibition against the albumin denaturation (83.50 ± 0.24%), whereas G. corticata was observed to measure a maximum inhibition in heat-induced hemolysis (60.40 ± 0.46%) and proteinase inhibition assay (83.30 ± 0.18%). An extensive literature survey was carried out for the bioactive compounds in G.corticata; it was examined for drug likeliness by ADME analysis and toxicological parameters. Further, the best selected bioactive compounds were subjected to in silico molecular docking with pro-inflammatory target, cyclooxygenase (COX-2). Hexadecanal and Neophytadiene were reported to obtain the highest binding affinity (-5.3) for COX-2 enzyme. Hence, in silico molecular docking studies had shown that G. corticata was found to possess potential anti-inflammatory activity that can prevent conversion of arachidonic acid to prostaglandins by inhibiting COX-2. In addition, molecular dynamic simulation studies have shown the stability of Hexadecanal-6COX complex. To conclude, the outcomes of the present study may shed light on the understanding of the usage of bioactive compounds for therapeutic purpose.

The role of CcPTGS2a in immune response against Aeromonas hydrophila infection in common carp (Cyprinus carpio)

Prostaglandin-endoperoxide synthase 2 (PTGS2), a crucial enzyme in prostaglandin synthesis, catalyzes the conversion of arachidonic acid to prostaglandins and plays a significant role in the inflammatory response. This investigation aimed to determine the regulatory role of PTGS2a in the innate immune response to bacterial infection in fish. To achieve this objective, the CcPTGS2a gene was identified and characterized in common carp (Cyprinus carpio), and its function in immune defense was investigated. According to the sequence and structural analysis results, CcPTGS2a had an open reading frame of 1806 bp that encoded 602 amino acids. It was estimated that the protein's theoretical molecular weight was 69.0 kDa, and its isoelectric point was 8.10. The structure of CcPTGS2a was observed to be conserved, with an epidermal growth factor domain and a peroxidase domain present. Moreover, the amino acid sequence of CcPTGS2a exhibited significant homology with the amino acid sequences of several fish species. CcPTGS2a mRNA was detected in the healthy tissues of common carp, with higher expression in the head kidney, spleen, gills, and liver. Following the challenges with Aeromonas hydrophila and lipopolysaccharide, CcPTGS2a mRNA showed unique geographic and temporal expression patterns, with significant increases detected in the head kidney, gills, spleen, and liver. Additionally, the recombinant CcPTGS2a protein exhibited detectable bacterial binding to various bacteria. As determined by subcellular localization analysis, CcPTGS2a was predominantly localized in the nucleus and cytoplasm. Furthermore, it was discovered that the overexpression of CcPTGS2a stimulated the up-regulation of ferroptosis-related genes and inflammatory cytokine mRNA expression in fish and EPC (Epithelioma papulosum cyprinid) cells while concurrently reducing the bacterial load of A. hydrophila. In contrast, the interference of CcPTGS2a decreased the mRNA expression of ferroptosis-related genes and inflammatory cytokines in fish and EPC cells and increased the bacterial load of A. hydrophila. Notably, A. hydrophila stimulation resulted in the up-regulation of CcPTGS2a protein expression in EPC cells. These results suggested that CcPTGS2a was involved in the immune response to bacterial infections in common carp.

To study the utility of COX-2 as immunohistochemical prognostic marker in comparison to various histopathological parameters and TNM staging in breast carcinoma: an observational, cross-sectional study protocol

Background: Breast cancer is the most prevalent cancer among women worldwide and is a well-known cause for cancer mortality in females. COX-2 (cyclooxygenase) plays a vital role in development of some human cancers such as lung, colon and breast. It is a potent enzyme that is important for the conversion of arachidonic acid into prostaglandins. These prostaglandins mediate cellular proliferation, apoptosis and angiogenesis which contributes to carcinogenesis. Overexpression of COX-2 has been detected in several malignancies including breast cancer. COX-2 overexpression is regarded as a poor prognostic marker of breast cancer. The present study will aim to study the immunohistochemical expression of COX-2 in breast cancer and compare it with known histopathological parameters thus assessing its prognostic value. Methods: This will be an observational study conducted in the Department of Pathology, JNMC, Wardha (Sawangi). Radical mastectomy specimens will be studied for COX-2 expression by immunohistochemistry in patients diagnosed with breast carcinoma. COX-2 expression will be quantified as immunohistochemical score and results will be correlated with various histopathological parameters. Results: The expected result of our study will suggest an association of COX-2 expression to the factors associated with poor prognosis in breast carcinoma. A positive correlation is expected between larger tumor size, positive lymph node status, higher T stage and N stage and lymphovascular invasion. Conclusions: Conclusions will be drawn from the obtained results of the immunohistochemical study by using COX-2- for detection of overexpression of COX-2 when evaluated with TNM staging, histological grading and molecular types of breast cancer.

Impact of Lipid Metabolism on Macrophage Polarization: Implications for Inflammation and Tumor Immunity.

Macrophage polarization is influenced by lipids, which also exert significant control over macrophage functions. Lipids and their metabolites are players in intricate signaling pathways that modulate macrophages' responses to pathogens, phagocytosis, ferroptosis, and inflammation. This review focuses on lipid metabolism and macrophage functions and addresses potential molecular targets for the treatment of macrophage-related diseases. While lipogenesis is crucial for lipid accumulation and phagocytosis in M1 macrophages, M2 macrophages likely rely on fatty acid β-oxidation to utilize fatty acids as their primary energy source. Cholesterol metabolism, regulated by factors such as SREBPs, PPARs, and LXRs, is associated with the cholesterol efflux capacity and the formation of foam cells (M2-like macrophages). Foam cells, which are targets for atherosclerosis, are associated with an increase in inflammatory cytokines. Lipolysis and fatty acid uptake markers, such as CD36, also contribute to the production of cytokines. Enhancing the immune system through the inhibition of lipid-metabolism-related factors can potentially serve as a targeted approach against tumor cells. Cyclooxygenase inhibitors, which block the conversion of arachidonic acid into various inflammatory mediators, influence macrophage polarization and have generated attention in cancer research.

Plausibility of Daphnia magna as an alternative experimental model to evaluate effects on eicosanoid synthesis

Eicosanoids play important roles in inflammation, allergy, fever, and immune responses. In the eicosanoid pathway, cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandins and is a crucial target of nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, toxicological studies on the eicosanoid pathway are important for drug discovery and the evaluation of adverse health outcomes due to environmental contaminants. However, experimental models are limited owing to concerns regarding ethical standards. Thus, new alternative models for evaluating toxic effects on the eicosanoid pathway must be developed. To this end, we adopted an invertebrate species, Daphnia magna, as an alternative model. D. magna was exposed to ibuprofen, a major NSAID, for 6 and 24 h. Transcription of eicosanoid-related genes (pla2, cox, pgd synthase, pgd2r2, ltb4dh, and lox) was analyzed by qPCR, eicosanoids (arachidonic acid, prostaglandin F2, dihydroxy prostaglandin F2, and 5-hydroxyeicosatetraenoate) were quantified by multiple reaction monitoring, and enzyme-linked immunosorbent assay was used to determine protein levels of arachidonic acid and prostaglandin E2 (PGE2). After 6 h of exposure, transcription of the pla2 and cox genes was downregulated. In addition, the whole-body level of arachidonic acid, an upstream of COX pathway, increased by over 1.5-fold. The levels of PGE2, a downstream of COX pathway, decreased after 24 h of exposure. According to our results, it is expected that the eicosanoid pathway might be conserved in D. magna, at least partially. This indicates the plausibility of D. magna as an alternative model for the screening of new drugs or chemical toxicity.

Discovery of andrographolide hit analog as a potent cyclooxygenase-2 inhibitor through consensus MD-simulation, electrostatic potential energy simulation and ligand efficiency metrics

Cyclooxygenase-2 (COX-2) is the key enzyme responsible for the conversion of arachidonic acid to prostaglandins that display pro-inflammatory properties and thus, it is a potential target protein to develop anti-inflammatory drugs. In this study, chemical and bio-informatics approaches have been employed to find a novel potent andrographolide (AGP) analog as a COX-2 inhibitor having better pharmacological properties than aspirin and rofecoxib (controls). The full amino acid sequenced human Alpha fold (AF) COX-2 protein (604AA) was selected and validated for its accuracy against the reported COX-2 protein structures (PDB ID: 5F19, 5KIR, 5F1A, 5IKQ and 1V0X) followed by multiple sequence alignment analysis to establish the sequence conservation. The systematic virtual screening of 237 AGP analogs against AF-COX-2 protein yielded 22 lead compounds based on the binding energy score (< − 8.0 kcal/mol). These were further screened out to 7 analogs by molecular docking analysis and investigated further for ADMET prediction, ligand efficiency metrics calculations, quantum mechanical analysis, MD simulation, electrostatic potential energy (EPE) docking simulation, and MM/GBSA. In-depth analysis revealed that AGP analog A3 (3-[2-[(1R,4aR,5R,6R,8aR)-6-hydroxy-5,6,8a-trimethyl-2-methylidene-3,4,4a,5,7,8-hexahydro-1H-naphthalen-1-yl]ethylidene]-4-hydroxyoxolan-2-one) forms the most stable complex with the AF-COX-2 showing the least RMSD value (0.37 ± 0.03 nm), a good number of hydrogen bonds (protein–ligand H-bond = 11, and protein H-bond = 525), minimum EPE score (− 53.81 kcal/mol), and lowest MM-GBSA before and after simulation (− 55.37 and − 56.25 kcal/mol, respectively) value compared to other analogs and controls. Thus, we suggest that the identified A3 AGP analog could be developed as a promising plant-based anti-inflammatory drug by inhibiting COX-2.

Design, synthesis and evaluation of 2-aryl quinoline derivatives against 12R-lipoxygenase (12R-LOX): Discovery of first inhibitor of 12R-LOX

The 12R-lipoxygenase (12R-LOX), a (non-heme) iron-containing metalloenzyme belonging to the lipoxygenase (LOX) family catalyzes the conversion of arachidonic acid (AA) to its key metabolites. Studies suggested that 12R-LOX plays a critical role in immune modulation for the maintenance of skin homeostasis and therefore can be considered as a potential drug target for psoriasis and other skin related inflammatory diseases. However, unlike 12-LOX (or 12S-LOX) the enzyme 12R-LOX did not receive much attention till date. In our effort, the 2-aryl quinoline derivatives were designed, synthesized and evaluated for the identification of potential inhibitors of 12R-hLOX. The merit of selection of 2-aryl quinolines was assessed by in silico docking studies of a representative compound (4a) using the homology model of 12R-LOX. Indeed, in addition to participating in H-bonding with THR628 and LEU635 the molecule formed a hydrophobic interaction with VAL631. The desired 2-aryl quinolines were synthesized either via the Claisen-Schmidt condensation followed by one-pot reduction-cyclization or via the AlCl3 induced heteroarylation or via the O-alkylation approach in good to high (82–95%) yield. When screened against human 12R-LOX (12R-hLOX) in vitro four compounds (e.g. 4a, 4d, 4e and 7b) showed encouraging (>45%) inhibition at 100 μM among which 7b and 4a emerged as the initial hits. Both the compounds showed selectivity towards 12R-hLOX over 12S-hLOX, 15-hLOX and 15-hLOXB and concentration dependent inhibition of 12R-hLOX with IC50 = 12.48 ± 2.06 and 28.25 ± 1.63 μM, respectively. The selectivity of 4a and 7b towards 12R-LOX over 12S-LOX was rationalized with the help of molecular dynamics simulations. The SAR (Structure-Activity Relationship) within the present series of compounds suggested the need of a o-hydroxyl group on the C-2 phenyl ring for the activity. The compound 4a and 7b (at 10 and 20 µM) reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes in a concentration dependent manner. Further, both compounds decreased the protein levels of Ki67 and the mRNA expression of IL-17A in the IMQ-induced psoriatic-like keratinocytes. Notably, 4a but not 7b inhibited the production of IL-6 and TNF-α in the keratinocyte cells. In the preliminary toxicity studies (i.e. teratogenicity, hepatotoxicity and heart rate assays) in zebrafish both the compounds showed low safety (<30 µM) margin. Overall, being the first identified inhibitors of 12R-LOX both 4a and 7b deserve further investigations.

Investigating DAGLβ expression and activity in human monocytes via proteomic and lipidomic profiling

Abstract Lipid-metabolizing enzymes are important targets for small-molecule inhibitor discovery as the regulation of inflammation responses have been linked to lipid metabolism cascades controlled by these enzymes. Non-steroidal anti-inflammatory drugs inhibit enzymes in the COX pathway preventing conversion of arachidonic acid (AA) into inflammation-inducing prostaglandins. An upstream precursor to AA is the endocannabinoid 2-arachidonoyl-glycerol (2-AG), which is biosynthesized from the cleavage of arachidonate-containing diacylglycerols (DAGs) by diacylglycerol lipase beta (DAGLβ). Previous work to identify DAGLβ’s role in inflammatory function led to the creation of KT109, a DAGLβ-specific 1,2,3-triazole urea inhibitor. Primary mouse macrophages treated with KT109 showed reductions in AA and 2-AG and an increase in arachidonate-containing DAGs, demonstrating that DAGLβ is key in proinflammatory lipid regulation and signaling. These studies focused on the effects of inhibiting mouse DAGLβ, but information regarding KT109 inhibition of human DAGLβ is still lacking. Herein, we applied activity-based protein profiling (ABPP) and quantitative proteomic and lipidomic methods to determine the effectivity of KT109 inhibition in human monocytic cell line THP1. Potency (IC 50values) for DAGLβ inhibition by KT109 in human THP1 cells were comparable to the magnitude of inhibition in mouse cells. Lipidomic analysis of KT109-treated monocytic and macrophage-differentiated THP1s did not demonstrate an accumulation of arachidonate-containing DAGs. These findings show a potential difference in the expression and activity of human DAGLβ when compared to mouse DAGLβ and additional targets of KT109 in human cells. Funding and disclosures: This work was supported by the National Institute on Drug Abuse grant no. R01 DA043571.

Anti-Inflammatory Activities of Arnica montana Planta Tota versus Flower Extracts: Analytical, In Vitro and In Vivo Mouse Paw Oedema Model Studies

Arnica montana is well known for its anti-inflammatory properties. While the anti-inflammatory activity of Arnica flowers (Arnicae flos) has been extensively studied, that of the whole plant (Arnicae planta tota) is less characterized. We compared the ability of Arnicae planta tota and Arnicae flos extracts to inhibit the pro-inflammatory NF-κB-eicosanoid pathway, using several in vitro and in vivo assays. We showed that Arnicae planta tota inhibited NF-κB reporter activation, with an IC50 of 15.4 μg/mL (vs. 52.5 μg/mL for Arnicae flos). Arnicae planta tota also inhibited LPS-induced expression of ALOX5 and PTGS2 genes in human differentiated macrophages. ALOX5 and PTGS2 encode the 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2) enzymes that initialize the conversion of arachidonic acid into leukotrienes and prostaglandins, respectively. Arnicae planta tota inhibited 5-LO and COX-2 enzymatic activity in vitro and in human primary peripheral blood cells, with lower IC50 compared to Arnicae flos. Finally, Arnicae planta tota applied topically reduced carrageenan-induced mouse paw oedema more efficiently than Arnicae flos. Altogether, Arnicae planta tota displayed a superior anti-inflammatory activity compared to Arnicae flos, suggesting that Arnicae-planta-tota-containing products might be more effective in alleviating the manifestations of acute inflammation than those based on Arnicae flos alone.

The DNA adenine methylase of Salmonella Enteritidis promotes their intracellular replication by inhibiting arachidonic acid metabolism pathway in macrophages

Macrophages can participate in immune responses by altering their metabolism, and play important roles in controlling bacterial infections. However, Salmonella Enteritidis can survive and proliferate in macrophages. After the deletion of DNA adenine methylase (Dam), the proliferation of Salmonella Enteritidis in macrophages decreased, the molecular mechanism is still unclear. After infecting macrophages with Salmonella Enteritidis wild type and dam gene deletion strains, intracellular metabolites were extracted and detected by non-targeted metabolomics and fatty acid targeted metabolomics. We found Dam had significant effects on arachidonic acid and related metabolic pathways in macrophages. The dam gene can promote the proliferation of Salmonella Enteritidis in macrophages by inhibiting the metabolic pathway of cytosolic phospholipase A2-mediated arachidonic acid production and conversion to prostaglandin E2 in macrophages, reducing the secretion of the pro-inflammatory factors IL-1β and IL-6. In addition, inhibition of arachidonic acid-related pathways in macrophages by Arachidonyl trifluoromethyl ketone could restore the proliferation of dam gene deletion strains in macrophages. This study explored the role of Dam in the process of Salmonella Enteritidis invading host cells from the perspective of host cell metabolism, and provides new insights into the immune escape mechanism of Salmonella Enteritidis.