RESUMO
The COVID-19 pandemic has highlighted the lack of effective, ready-to-use antivirals for the treatment of viruses with pandemic potential. The development of a diverse drug portfolio is therefore crucial for pandemic preparedness. Viral macrodomains are attractive therapeutic targets as they are suggested to play an important role in evading the innate host immune response, making them critical for viral pathogenesis. Macrodomains function as erasers of mono-ADP-ribosylation (deMARylation), a post-translational modification that is involved in interferon signaling. Herein, we report the development of a modular HTRF-based assay, that can be used to screen for inhibitors of various viral and human macrodomains. We characterized the five most promising small molecule SARS-CoV-2 Mac1 inhibitors recently reported in the literature for potency and selectivity and conducted a pilot screen demonstrating HTS suitability. The ability to directly detect enzymatic activity makes the DeMAR assay a valuable addition to the existing tools for macrodomain drug discovery.
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G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure-activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.
Assuntos
Receptores Acoplados a Proteínas G , Humanos , Relação Estrutura-Atividade , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Animais , Fenilalanina/farmacologia , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/síntese química , Estrutura MolecularRESUMO
5-Lipoxygenase (5-LO), a fatty acid oxygenase, is the central enzyme in leukotriene (LT) biosynthesis, potent arachidonic acid-derived lipid mediators released by innate immune cells, that control inflammatory and allergic responses. In addition, through interaction with 12- and 15-lipoxgenases, the enzyme is involved in the formation of omega-3 fatty acid-based oxylipins, which are thought to be involved in the resolution of inflammation. The expression of 5-LO is frequently deregulated in solid and liquid tumors, and there is strong evidence that the enzyme plays an important role in carcinogenesis. However, global inhibition of LT formation and signaling has not yet shown the desired success in clinical trials. Curiously, the release of 5-LO-derived lipid mediators from tumor cells is often low, and the exact mechanism by which 5-LO influences tumor cell function is poorly understood. Recent data now show that in addition to releasing oxylipins, 5-LO can also influence gene expression in a lipid mediator-independent manner. These non-canonical functions, including modulation of miRNA processing and transcription factor shuttling, most likely influence cancer cell function and the tumor microenvironment and might explain the low clinical efficacy of pharmacological strategies that previously only targeted oxylipin formation and signaling by 5-LO. This review summarizes the canonical and non-canonical functions of 5-LO with a particular focus on tumorigenesis, highlights unresolved issues, and suggests future research directions.
Assuntos
Araquidonato 5-Lipoxigenase , Carcinogênese , Neoplasias , Animais , Humanos , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/genética , Carcinogênese/metabolismo , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Leucotrienos/metabolismo , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Transdução de SinaisRESUMO
Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.
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Canais de Potássio , Prurido , Animais , Camundongos , Antipruriginosos/uso terapêutico , Histamina/metabolismo , Loxapina/uso terapêutico , Canais de Potássio/metabolismo , Prurido/tratamento farmacológico , Prurido/metabolismoRESUMO
Ferroptosis is a form of oxidative cell death that is characterized by enhanced lipid peroxidation and mitochondrial impairment. The enzymes acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase (LPCAT) play an essential role in the biosynthesis of polyunsaturated fatty acid (PUFA)-containing phospholipids, thereby providing the substrates for lipid peroxidation and promoting ferroptosis. To examine the impact of mitochondria in ACSL4/LPCAT2-driven ferroptosis, HEK293T cells overexpressing ACSL4 and LPCAT2 (OE) or empty vector controls (LV) were exposed to 1S, 3R-RSL3 (RSL3) for induction of ferroptosis. The ACSL4/LPCAT2 overexpression resulted in higher sensitivity against RSL3-induced cell death compared to LV-transfected controls. Moreover, mitochondrial parameters such as mitochondrial reactive oxygen species (ROS) formation, mitochondrial membrane potential, and mitochondrial respiration deteriorated in the OE cells, supporting the conclusion that mitochondria play a significant role in ACSL4/LPCAT2-driven ferroptosis. This was further confirmed through the protection of OE cells against RSL3-mediated cell death by the mitochondrial ROS scavenger mitoquinone (MitoQ), which exerted protection via antioxidative properties rather than through previously reported metabolic effects. Our findings implicate that mitochondrial ROS production and the accompanying organelle disintegration are essential for mediating oxidative cell death initiated through lipid peroxidation in ferroptosis.
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Nitro-fatty acids (NFAs) are endogenous lipid mediators causing a spectrum of anti-inflammatory effects by covalent modification of key proteins within inflammatory signaling pathways. Recent animal models of solid tumors have helped demonstrate their potential as anti-tumorigenic therapeutics. This study evaluated the anti-tumorigenic effects of NFAs in colon carcinoma cells and other solid and leukemic tumor cell lines. NFAs inhibited the ubiquitin-proteasome system (UPS) by directly targeting the 26S proteasome, leading to polyubiquitination and inhibition of the proteasome activities. UPS suppression induced the unfolded protein response, resulting in tumor cell death. The NFA-mediated effects were substantial, specific, and enduring, representing a unique mode of action for UPS suppression. This study provides mechanistic insights into the biological actions of NFAs as possible endogenous tumor-suppressive factors, indicating that NFAs might be key structures for designing a novel class of direct proteasome inhibitors.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ácidos Graxos/farmacologia , Inibidores de Proteassoma/farmacologiaRESUMO
Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di-tert-butylphenol derivatives R-830, S-2474, KME-4, and E-5110 represent potent dual COX-2/5-LO inhibitors. The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that showed no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibited lower anticancer activity compared to the related di-tert-butylphenols. Interestingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell proliferation more potently than its carbon-based R-830 counterpart. Considering all the advantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.
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Boranos , Inibidores de Lipoxigenase , Humanos , Ciclo-Oxigenase 2 , Inibidores de Lipoxigenase/farmacologiaRESUMO
A library of 43 thiazole derivatives, including 31 previously and 12 newly synthesized in the present study, was evaluated in vitro for their inhibitory properties against bovine pancreatic DNase I. Nine compounds (including three newly synthesized) inhibited the enzyme showing improved inhibitory properties compared to that of the reference crystal violet (IC50 = 346.39 µM). Two compounds (5 and 29) stood out as the most potent DNase I inhibitors, with IC50 values below 100 µM. The 5-LO inhibitory properties of the investigated derivatives were also analyzed due to the importance of this enzyme in the development of neurodegenerative diseases. Compounds (12 and 29) proved to be the most prominent new 5-LO inhibitors, with IC50 values of 60 nM and 56 nM, respectively, in cell-free assay. Four compounds, including one previously (41) and three newly (12, 29 and 30) synthesized, have the ability to inhibit DNase I with IC50 values below 200 µM and 5-LO with IC50 values below 150 nM in cell-free assay. Molecular docking and molecular dynamics simulations were used to clarify DNase I and 5-LO inhibitory properties of the most potent representatives at the molecular level. The newly synthesized compound 29 (4-((4-(3-bromo-4-morpholinophenyl)thiazol-2-yl)amino)phenol) represents the most promising dual DNase I and 5-LO inhibitor, as it inhibited 5-LO in the nanomolar and DNase I in the double-digit micromolar concentration ranges. The results obtained in the present study, together with our recently published results for 4-(4-chlorophenyl)thiazol-2-amines, represent a good basis for the development of new neuroprotective therapeutics based on dual inhibition of DNase I and 5-LO.
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Fármacos Neuroprotetores , Tiazóis , Animais , Bovinos , Relação Estrutura-Atividade , Tiazóis/farmacologia , Tiazóis/química , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Araquidonato 5-Lipoxigenase , Desoxirribonuclease I , Inibidores de Lipoxigenase/farmacologia , Estrutura MolecularRESUMO
The presence of inflammatory mediators in the tumor microenvironment, such as cytokines, growth factors or eicosanoids, indicate cancer-related inflammatory processes. Targeting these inflammatory mediators and related signal pathways may offer a rational strategy for the treatment of cancer. This study focuses on the incorporation of metabolically stable, sterically demanding, and hydrophobic dicarba-closo-dodecaboranes (carboranes) into dual cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids. The di-tert-butylphenol derivative tebufelone represents a selective dual COX-2/5-LO inhibitor. The incorporation of meta- or para-carborane into the tebufelone scaffold resulted in eight carborane-based tebufelone analogs that show no COX inhibition but 5-LO inhibitory activity inâ vitro. Cell viability studies on HT29 colon adenocarcinoma cells revealed that the observed antiproliferative effect of the para-carborane analogs of tebufelone is enhanced by structural modifications that include chain elongation in combination with introduction of a methylene spacer resulting in higher anticancer activity compared to tebufelone. Hence, this strategy proved to be a promising approach to design potent 5-LO inhibitors with potential application as cytostatic agents.
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Adenocarcinoma , Boranos , Neoplasias do Colo , Humanos , Ciclo-Oxigenase 2/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/química , Microambiente TumoralRESUMO
Specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins are formed by the consecutive action of 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- or 15-lipoxygenases using arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid as substrate. Lipoxins are trihydroxylated oxylipins which are formed from arachidonic and eicosapentaenoic acid. The latter can also be converted to di- and trihydroxylated resolvins of the E series, whereas docosahexaenoic acid is the substrate for the formation of di- and trihydroxylated resolvins of the D series. Here, we summarize the formation of lipoxins and resolvins in leukocytes. From the data published so far, it becomes evident that FLAP is required for the biosynthesis of most of the lipoxins and resolvins. Even in the presence of FLAP, formation of the trihydroxylated SPMs (lipoxins, RvD1-RvD4, RvE1) in leukocytes is very low or undetectable which is obviously due to the extremely low epoxide formation by 5-LO from oxylipins such as 15-H(p)ETE, 18-H(p)EPE or 17-H(p)DHA. As a result, only the dihydroxylated oxylipins (5 S,15S-diHETE, 5 S,15S-diHEPE) and resolvins (RvD5, RvE2, RvE4) can be consistently detected using leukocytes as SPM source. However, the reported levels of these dihydroxylated lipid mediators are still much lower than those of the typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g. 5-HETE), leukotrienes or cyclooxygenase-derived prostaglandins. Since 5-LO expression is mainly restricted to leukocytes these cells are considered as the main source of SPMs. The low formation of trihydroxylated SPMs in leukocytes, the fact that they are hardly detected in biological samples as well as the lack of functional signaling by their receptors make it highly questionable that trihydroxylated SPMs play a role as endogenous mediators in the resolution of inflammation.
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Lipoxinas , Humanos , Lipoxinas/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico , Ácido Araquidônico , Oxilipinas , Eicosanoides/metabolismo , Inflamação/metabolismo , LeucócitosRESUMO
Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.
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Cisteína , Prostaglandinas , Camundongos , Humanos , Animais , Lipopolissacarídeos/metabolismo , Mastócitos , Prostaglandina-E Sintases/metabolismo , Macrófagos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Prostaglandina D2/farmacologiaRESUMO
Human 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis, mostly expressed in leukocytes and thus a crucial component of the innate immune system. In this study, we show that 5-LO, besides its canonical function as an arachidonic acid metabolizing enzyme, is a regulator of gene expression associated with euchromatin. By Crispr-Cas9-mediated 5-LO knockout (KO) in MonoMac6 (MM6) cells and subsequent RNA-Seq analysis, we identified 5-LO regulated genes which could be clustered to immune/defense response, cell adhesion, transcription and growth/developmental processes. Analysis of differentially expressed genes identified cyclooxygenase-2 (COX2, PTGS2) and kynureninase (KYNU) as strongly regulated 5-LO target genes. 5-LO knockout affected MM6 cell adhesion and tryptophan metabolism via inhibition of the degradation of the immunoregulator kynurenine. By subsequent FAIRE-Seq and 5-LO ChIP-Seq analyses, we found an association of 5-LO with euchromatin, with prominent 5-LO binding to promoter regions in actively transcribed genes. By enrichment analysis of the ChIP-Seq results, we identified potential 5-LO interaction partners. Furthermore, 5-LO ChIP-Seq peaks resemble patterns of H3K27ac histone marks, suggesting that 5-LO recruitment mainly takes place at acetylated histones. In summary, we demonstrate a noncanonical function of 5-LO as transcriptional regulator in monocytic cells.
Assuntos
Araquidonato 5-Lipoxigenase , Eucromatina , Araquidonato 15-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Eucromatina/genética , Histonas/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipoxigenase/genética , Lipoxigenase/metabolismoRESUMO
Focused compound libraries are well-established tools for hit identification in drug discovery and chemical probe development. We present the compilation and application of a focused screening library of fatty acid mimetics (FAMs), which are compounds designed to bind the orthosteric site of proteins that endogenously accommodate natural fatty acids and lipid metabolites. This set complies with chemical properties of FAM and was found suitable for use also in cellular setting. Several hits were retrieved in screening the focused library against diverse fatty acid binding targets including the enzymes soluble epoxide hydrolase (sEH) and leukotriene A4 hydrolase (LTA4H), the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), the carrier proteins fatty acid binding protein 4 and 5 (FABP4 and FABP5), as well as the G-protein coupled receptors leukotriene B4 receptor 1 (BLT1) and free-fatty acid receptor 1 (FFAR1). Thus, the focused FAM library is suitable to obtain chemical starting matter for fatty acid binding proteins and provides a valuable extension to available screening collections.
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Epóxido Hidrolases , Ácidos Graxos , Epóxido Hidrolases/metabolismo , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , PPAR gama/metabolismo , Receptores do Leucotrieno B4/metabolismo , Receptor X Retinoide alfa/metabolismoRESUMO
Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.