Maria Grazia Perronea, Morena Miciaccia, S. Ferorelli, A. Scilimati
{"title":"A simplified direct O2 consumption-based assay to test COX inhibition","authors":"Maria Grazia Perronea, Morena Miciaccia, S. Ferorelli, A. Scilimati","doi":"10.2174/1573408018666220204104612","DOIUrl":"https://doi.org/10.2174/1573408018666220204104612","url":null,"abstract":"\u0000\u0000Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA) producing the hydroperoxide PGG2, then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions are a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors.\u0000\u0000\u0000\u0000Methods: we developed a new simplified and easy to be carried out protocol for O2 consumption measurement by using disrupted HEK293-derived adherent cells, stably transfected either with COX-1 or COX-2 genes, as a source of the COX enzymes. The Clark electrode is used to measure the O2 concentration variation during the enzyme-catalyzed reactions.\u0000\u0000\u0000\u0000Results and Discussion: the novel assay was validated by determining the IC50 values of the known inhibitors such as indomethacin, ibuprofen, SC560 and celecoxib. Indomethacin and ibuprofen are two traditional non-steroidal anti-inflammatory drugs (NSAIDs). SC560 is a commercially available reference compound used for COX-1 inhibition investigations. Celecoxib is a clinically used COXIBs. The assay was also applied to measure the kinetics and IC50 of mofezolac and P6. Mofezolac is the most potent selective COX-1 inhibitor and active principal ingredient of Disopain® used to treat the rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as reference in “in vitro” and “in vivo” COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies. The obtained results showed the suitability of the newly developed assay to measure COXs activity in the presence of inhibitors as well as the kinetics of the inhibition (i.e., Vmax and Km).\u0000","PeriodicalId":35405,"journal":{"name":"Current Enzyme Inhibition","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42202110","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
J. Piermattey, Maicol Ahumedo, Y. Heuze, Juan Soriano, Marisa Salinas
{"title":"New nonsteroidal molecules as blockers of the steroidogenic pathway","authors":"J. Piermattey, Maicol Ahumedo, Y. Heuze, Juan Soriano, Marisa Salinas","doi":"10.2174/1573408018666220106151712","DOIUrl":"https://doi.org/10.2174/1573408018666220106151712","url":null,"abstract":"\u0000\u0000Background: Testosterone circulating levels decrease in aging. This fact affects the emotional response to captivating pictures. Therefore, naturally increasing androgens within neurons could be a way to improve the mood of agedpeople.\u0000\u0000\u0000\u0000This study aimed to determine the biological activity of new nonsteroidal derivatives of 2-aminonaphthalene-1,4-dione (2-amino-3-iodonaphthalene-1,4-dione and 2-(iodoamino)-3-methylnaphthalene-1,4-dione) as inhibitors of the aldo-keto reductase 1 enzymes (AKR1C1, AKR1C2).\u0000\u0000\u0000\u0000The 2-amino-3-iodonaphthalene-1,4-dione and 2-(iodoamino)-3-methylnaphthalene-1,4-dione were synthesized, and their effect in vivo and in vitro was determined. The human prostate cell membrane was used as a source of steroidogenic enzymes. The 2-amino-3-iodonaphthalene-1,4-dione and 2-(iodoamino)-3-methylnaphthalene-1,4-dione bindings to the androgen receptors were also assayed using cytosol from the rat prostate. In vivo experiments, we determined the effects of 2-amino-3-iodonaphthalene-1,4-dione, 2-(iodoamino)-3-methylnaphthalene-1,4-dione on the weight of androgen-dependent glands of castrated hamsters treated with testosterone and finasteride or 2-amino-3-iodonaphthalene-1,4-dione or 2-(iodoamino)-3-methylnaphthalene-1,4-dione was determined.\u0000\u0000\u0000\u00002-amino-3-iodonaphthalene-1,4-dione and 2-(iodoamino)-3-methylnaphthalene-1,4-dione inhibited AKR1C1 enzyme activity with an IC50 value of 420 nM (2-amino-3-iodonaphthalene-1,4-dione) and 1.95 µM (2-(iodoamino)-3-methylnaphthalene-1,4-dione), respectively. They also blocked AKR1C2 with an IC50 value of 300 nM (2-amino-3-iodonaphthalene-1,4-dione) and 1.52 µM (2-(iodoamino)-3-methylnaphthalene-1,4-dione). Thus 2-amino-3-iodonaphthalene-1,4-dione and 2-(iodoamino)-3-methylnaphthalene-1,4-dione prevent the formation of 3α and 3β-androstanediols. Moreover, these compounds did not bind to AR and did not reduce prostate and seminal vesicle weight. The latter is because of the accumulation of dihydrotestosterone, which is an anabolic androgen.\u0000\u0000\u0000\u00002-amino-3-iodonaphthalene-1,4-dione and 2-(iodoamino)-3-methylnaphthalene-1,4-dione inhibited AKR1C1 and AKR1C2 enzyme activity; consequently, dihydrotestosterone was accumulated in androgen-dependent glands. These derivatives could potentially use therapeutics via direct nasal administration in aged patients, increasing DHT in neurons.\u0000","PeriodicalId":35405,"journal":{"name":"Current Enzyme Inhibition","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48481417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Allaoua Nouri, Lakhder Gasmi, Chawki Bensebini, D. Harzallah, S. Khennouf, S. Dahamna
{"title":"The Inhibitory effect of hydroalcoholic extract from the Algerian Echium trygorrhizum Pomel roots on α-amylase activity.","authors":"Allaoua Nouri, Lakhder Gasmi, Chawki Bensebini, D. Harzallah, S. Khennouf, S. Dahamna","doi":"10.2174/1573408018666211224092636","DOIUrl":"https://doi.org/10.2174/1573408018666211224092636","url":null,"abstract":"\u0000\u0000Species of Echium trygorrhizum Pomel , are used traditionally in Algeria folk medicine for the treatment of Diabetes, Jaundice and Tonsillitis. To our knowledge, no previous study has been conducted out on the pharmacological activities of this species. \u0000\u0000\u0000\u0000The objective of the present research was to evaluate the content of polyphenols , flavonoids and condensed tannins compounds and to assess in vitro the antioxidant activity and the inhibitory effect of the hydroalcoholic extract of this plant, on α-amylase activity, an enzyme responsible for digestion of carbohydrate before the process of intestinal absorption.\u0000\u0000\u0000\u0000Polyphenols, flavonoids and condensed tannins were evaluated spectrophotometrically using Folin–Ciocalteu, the Aluminum chloride and vanillin methods respectively. The antioxidant activity using DPPH radical scavenging, ABTS, Ferric reducing antioxidant power and β- carotene bleaching tests and the assessment of in vitro α- amylase inhibitory potential by an Aspergillus oryzae α- amylase have been studied.\u0000\u0000\u0000\u0000The hydroalcoholic crude extract was able to inhibit the α-amylase enzyme in vitro, with an IC50 of 0.56 ± 0.044 mg/ml, in addition the contents of polyphenols and flavonoids were found to be 157.403 ± 0.835 µg GAE/mg extract and 30.156±2.634µg Q E / mg extract, respectively, Whereas the amount of tannins was 65.293 ± 0.883µg Cat E/ mg of dry extract.\u0000\u0000\u0000\u0000The present study revealed that the extract is rich in phenolic compounds, which play a really important role in the scavenging of free radicals. It has been reported the inhibitory capacity of hydroalcoholic roots extract on Aspergillus oryzae α-amylase enzyme might be used as a natural agent within the management of diabetes mellitus. \u0000","PeriodicalId":35405,"journal":{"name":"Current Enzyme Inhibition","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45911761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Morena Miciaccia, M. Iaselli, S. Ferorelli, P. L. Polosa, M. Perrone, A. Scilimati
{"title":"Ovine COX-1 isoenzyme bio-production","authors":"Morena Miciaccia, M. Iaselli, S. Ferorelli, P. L. Polosa, M. Perrone, A. Scilimati","doi":"10.2174/1573408017666211108104731","DOIUrl":"https://doi.org/10.2174/1573408017666211108104731","url":null,"abstract":"\u0000\u0000Recent findings enlightened the pivotal role of cyclooxygenases-1 and -2 (COX-1 and COX-2) in human diseases with inflammation as the committed earliest stage, such as cancer and neurodegenerative diseases. COXs are the main targets of nonsteroidal anti-inflammatory drugs and catalyze the bis-oxygenation of arachidonic acid into prostaglandin PGH2, then converted into prostaglandins, thromboxane, and prostacyclin by tissue-specific isomerases. A remarkable amount of pure COX-1 results is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. \u0000\u0000\u0000\u0000\u0000 Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. \u0000\u0000\u0000\u0000\u0000 An improvement of a previously reported method for pure recombinant oCOX-1 production and isolation has been achieved, leading to a lower starting volume of infected cells for each purification, an increased cell density, and of the number of viral particles per cell, and a shortened infection period. The protocol for the recombinant oCOX-1 expression and purification has been in-depth elaborated to obtain 1 mg/L of protein.\u0000\u0000\u0000\u0000\u0000The optimized procedure could be suitable for producing other membrane proteins as well, for which an improvement in the solubilization step is necessary to have the availability of high concentration proteins.\u0000\u0000","PeriodicalId":35405,"journal":{"name":"Current Enzyme Inhibition","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44892538","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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