Current Enzyme Inhibition - Volume 18, Issue 1, 2022

Background: Recent 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 is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. Methods: Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine(o)COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. Results and Discussion: 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, an increased 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. Conclusion: The 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.

Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA), producing the hydroperoxide Prostaglandin G2 (PGG2), then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions is a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors. Methods: 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. Results 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 (tNSAIDs). 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 principle ingredient of Disopain® used to treat rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as a reference in in vitro and in vivo COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies. Conclusion: 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).

Background: 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 aged people. Objective: This 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). Methods: The 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 androgendependent 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. Results: 2-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. Conclusion: 2-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.

Background: Epilepsy is a constant neurological disorder influencing around 50 million individuals globally. However, most epileptic patients do not react to accessible medications and clinical treatments. This research work has been planned to produce novel 1,5-benzodiazepines from chalcone intermediates by ultrasonic irradiation method and perform the anticonvulsant activity by pentylenetetrazole incited seizures tests. Methods: Chalcones are used as precursors for synthesizing 1, 5-benzodiazepines by a reaction with ophenylenediamine in absolute ethanol in the presence of glacial acetic acid as a catalyst. The proposed synthesized structures were characterized by melting point, TLC, FTIR, ¹H & ¹³CNMR, and mass spectroscopy. All the molecules were assessed for anticonvulsant activity. Results: Anticonvulsant activity uncovered the fact that the mixes derived from dimethylamino, dimethoxy, hydroxy-substituted chalcones in the 1, 5-benzodiazepines can be used for seizures in mice. These pharmacological examinations have shown that these new subsidiaries can repress seizures incited by pentylenetetrazole in mice proficiently. Our molecular docking studies also supported probable effects. Conclusion: The results are promising, which on further assessments may provoke medicine particles against seizures in mice.

Background: Species 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 on the pharmacological activities of this species. Objective: The 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. Methods: Polyphenols, 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. Results: The 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. Conclusion: The present study revealed that the extract is rich in phenolic compounds, which play a really important role in the scavenging of free radicals. The inhibitory capacity of hydroalcoholic roots extract on Aspergillus oryzae α-amylase enzyme has been reported and might be used as a natural agent within the management of diabetes mellitus.

Background: Diabetes has a large death toll worldwide, particularly as it falls into the ten leading causes of death. Type 2 diabetes mellitus (T2DM) occurs as the body becomes resistant to insulin and sugar accumulates in the blood. It has been observed that dipeptidyl peptidase-IV (DPP-IV) inhibitors and glucokinase activators are known therapeutic agents to treat T2DM. Among the possible medicinal plants, Gymnema sylvestre (GyS) belongs to the Apocynaceae family and is traditionally used for the treatment of different diseases. This plant is also known as “Gurmur” because it has a sugarreducing ability. GyS is known to be one of the main botanicals for the treatment of diabetes. Objective: Considering the studies described above, we have tried to investigate the natural DPP-IV inhibitors and potent glucokinase activators from the phytoconstituents of GyS. New drug candidates from the medicinal plant GyS have been reported as potent DPP-IV inhibitors and glucokinase activators. Methods: As a preliminary investigation, we have studied the effectiveness of phytoconstituents of GyS in T2DM through molecular docking as a proof of concept of synthesizing silver nanoparticles (for the treatment of T2DM) using an extract of this plant. Results: The present investigative research shows that the recognized compounds included in the present analysis have important values in the treatment of diabetes mellitus. The nine compounds selected are evaluated on the basis of DPP-IV and glucokinase enzyme binding energy values and their drug properties. Except for quercitol, all the selected compounds have exhibited much more potent glucokinase activation potential than their native ligands. Gymnemasin A, lupeol, gymnemoside A, gymnemasaponin V, and gymnemic acid I have shown excellent DPP-IV inhibitory potential. Conclusion: We aimed to synthesize the silver nanoparticles from the leaf extract of GyS for the treatment of T2DM. As a preliminary investigation, we have studied the effectiveness of phytoconstituents of GyS in T2DM through molecular docking as proof of synthesizing silver nanoparticles (for the treatment of T2DM) using an extract of this plant. As a result of the present investigation, it has been concluded that these compounds can be used to treat T2DM, and hence, in the future, we can synthesize the silver nanoparticles from the GyS extract for the treatment of T2DM.

Background: Glucokinase (GK) is a cytoplasmic enzyme that metabolizes glucose to glucose- 6-phosphate and supports adjusting blood glucose levels within the normal range in humans. In pancreatic β-cells, it plays a leading role in governing the glucose-stimulated secretion of insulin, and in liver hepatocyte cells, it controls the metabolism of carbohydrates. GK acts as a promising drug target for treating patients with type 2 diabetes mellitus (T2DM). Objectives: The present work has been designed to discover some novel substituted benzamide derivatives. Methods: This work involved designing novel benzamide derivatives and their screening by docking studies to determine the binding interactions for the best-fit conformations in the binding site of the GK enzyme. Based on the results of docking studies, the selected molecules were synthesized and tested for in vitro GK enzyme assay. The structures of newly synthesized products were confirmed by IR, NMR, and mass spectroscopy. Results: Amongst the designed derivatives, compounds 4c, 4d, 4e, 5h, 5j, 5l, 5m, 5n, 5p, and 5r have shown better binding energy than the native ligand present in the enzyme structure. The synthesized compounds were subjected to in vitro GK enzyme assay. Out of all, compounds 4c, 4d, 5h, 5l, and 5n showed more GK activation than control. Conclusion: From the present results, we have concluded that the synthesized derivatives can activate the human GK enzyme effectively, which can be helpful in the treatment of T2DM.