Microbiology & Cell Biology
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Item Evaluation of Nitric Oxide-Donating Properties of 11H-indeno[1,2-b]quinoxalin-11-one Oxime (IQ-1) by Electron Paramagnetic Resonance Spectroscopy(MDPI AG, 2024-08) Andrianov, Viacheslav V.; Schepetkin, Igor A.; Bazan, Leah V.; Gainutdinov, Khalil L.; Kovrizhina, Anastasia R.; Atochin, Dmitriy N.; Khlebnikov, Andrei I.IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) is a specific c-Jun N-terminal kinase (JNK) inhibitor with anticancer and neuro- and cardioprotective properties. Because aryloxime derivatives undergo cytochrome P450-catalyzed oxidation to nitric oxide (NO) and ketones in liver microsomes, NO formation may be an additional mechanism of IQ-1 pharmacological action. In the present study, electron paramagnetic resonance (EPR) of the Fe2+ complex with diethyldithiocarbamate (DETC) as a spin trap and hemoglobin (Hb) was used to detect NO formation from IQ-1 in the liver and blood of rats, respectively, after IQ-1 intraperitoneal administration (50 mg/kg). Introducing the spin trap and IQ-1 led to signal characteristics of the complex (DETC)2-Fe2+-NO in rat liver. Similarly, the introduction of the spin trap components and IQ-1 resulted in an increase in the Hb-NO signal for both the R- and the T-conformers in blood samples. The density functional theory (DFT) calculations were in accordance with the experimental data and indicated that the NO formation of IQ-1 through the action of superoxide anion radical is thermodynamically favorable. We conclude that the administration of IQ-1 releases NO during its oxidoreductive bioconversion in vivo.Item Phytochemical Composition and Biological Activity of the Essential Oil from Ericameria nauseosa Collected in Southwestern Montana, United States(MDPI AG, 2024-07) Schepetkin, Igor A.; Özek, Gulmira; Özek, Temel; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Ayçiçek, Kevser; Lavin, Matthew; Quinn, Mark T.Ericameria nauseosa (Pall. ex Pursh) G.L. Nesom & G.I. Baird) is used in traditional medicine to treat various diseases; however, little is known about the immunomodulatory activity of essential oil from this plant. Thus, we isolated essential oil from the aerial parts of E. nauseosa and evaluated their chemical composition and biological activity. Compositional analysis of E. nauseosa essential oil revealed that the main (>2%) components were γ-decalactone (13.3%), cryptone (9.4%), terpinen-4-ol (9.3%), (E)-methyl cinnamate (6.0%), T-cadinol (4.7%), spathulenol (3.6%), 8Z-2,3-dihydromatricaria ester (3.1%), β-phellandrene (3.0%), p-cymen-8-ol (2.2%), 3-ethoxy-2-cycloocten-1-one (2.2%), and trans-p-menth-2-en-1-ol (2.1%). Distinctive features were the lactones (up to 15%) and polyacetylenes (up to 3.1%), including (2Z,8Z)-matricaria ester and 8Z-2,3-dihydromatricaria ester. A comparison with other reported E. nauseosa essential oil samples showed that our samples were distinct from those collected in other areas of the country; however, they did have the most similarity to one sample collected in North Central Utah. Pharmacological studies showed that E. nauseosa essential oil activated human neutrophil Ca2+ influx, which desensitized these cells to subsequent agonist-induced functional responses. Based on our previously reported data that nerolidol, β-pinene, spathulenol, sabinene, and γ-terpinene were active in human neutrophils, these compounds are the most likely constituents contributing to this immunomodulatory activity. However, the relatively high amount of polyacetylenes may also contribute, as these compounds have been characterized as potent immunomodulators.Item Anti-Inflammatory Activity of Pyrazolo[1,5-a]quinazolines(MDPI AG, 2024-05) Crocetti, Letizia; Khlebnikov, Andrei I.; Guerrini, Gabriella; Schepetkin, Igor A.; Melani, Fabrizio; Giovannoni, Maria Paola; Quinn, Mark T.Chronic inflammation contributes to a number of diseases. Therefore, control of the inflammatory response is an important therapeutic goal. To identify novel anti-inflammatory compounds, we synthesized and screened a library of 80 pyrazolo[1,5-a]quinazoline compounds and related derivatives. Screening of these compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP-1Blue monocytic cells identified 13 compounds with anti-inflammatory activity (IC50 < 50 µM) in a cell-based test system, with two of the most potent being compounds 13i (5-[(4-sulfamoylbenzyl)oxy]pyrazolo[1,5-a]quinazoline-3-carboxamide) and 16 (5-[(4-(methylsulfinyl)benzyloxy]pyrazolo[1,5-a]quinazoline-3-carboxamide). Pharmacophore mapping of potential targets predicted that 13i and 16 may be ligands for three mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2), p38α, and c-Jun N-terminal kinase 3 (JNK3). Indeed, molecular modeling supported that these compounds could effectively bind to ERK2, p38α, and JNK3, with the highest complementarity to JNK3. The key residues of JNK3 important for this binding were identified. Moreover, compounds 13i and 16 exhibited micromolar binding affinities for JNK1, JNK2, and JNK3. Thus, our results demonstrate the potential for developing lead anti-inflammatory drugs based on the pyrazolo[1,5-a]quinazoline and related scaffolds that are targeted toward MAPKs.Item Volatile Composition, Antimicrobial Activity, and In Vitro Innate Immunomodulatory Activity of Echinacea purpurea (L.) Moench Essential Oils(MDPI AG, 2023-10) Dosoky, Noura S.; Kirpotina, Liliya N.; Schepetkin, Igor A.; Khlebnikov, Andrei I.; Lisonbee, Brent L.; Black, Jeffrey L.; Woolf, Hillary; Thurgood, Trever L.; Graf, Brittany L.; Satyal, Prabodh; Quinn, Mark T.Echinacea purpurea (L.) Moench is a medicinal plant commonly used for the treatment of upper respiratory tract infections, the common cold, sore throat, migraine, colic, stomach cramps, and toothaches and the promotion of wound healing. Based on the known pharmacological properties of essential oils (EOs), we hypothesized that E. purpurea EOs may contribute to these medicinal properties. In this work, EOs from the flowers of E. purpurea were steam-distilled and analyzed by gas chromatography–mass spectrometry (GC–MS), GC with flame-ionization detection (GC–FID), and chiral GC–MS. The EOs were also evaluated for in vitro antimicrobial and innate immunomodulatory activity. About 87 compounds were identified in five samples of the steam-distilled E. purpurea EO. The major components of the E. purpurea EO were germacrene D (42.0 ± 4.61%), α-phellandrene (10.09 ± 1.59%), β-caryophyllene (5.75 ± 1.72%), γ-curcumene (5.03 ± 1.96%), α-pinene (4.44 ± 1.78%), δ-cadinene (3.31 ± 0.61%), and β-pinene (2.43 ± 0.98%). Eleven chiral compounds were identified in the E. purpurea EO, including α-pinene, sabinene, β-pinene, α-phellandrene, limonene, β-phellandrene, α-copaene, β-elemene, β-caryophyllene, germacrene D, and δ-cadinene. Analysis of E. purpurea EO antimicrobial activity showed that they inhibited the growth of several bacterial species, although the EO did not seem to be effective for Staphylococcus aureus. The E. purpurea EO and its major components induced intracellular calcium mobilization in human neutrophils. Additionally, pretreatment of human neutrophils with the E. purpurea EO or (+)-δ-cadinene suppressed agonist-induced neutrophil calcium mobilization and chemotaxis. Moreover, pharmacophore mapping studies predicted two potential MAPK targets for (+)-δ-cadinene. Our results are consistent with previous reports on the innate immunomodulatory activities of β-caryophyllene, α-phellandrene, and germacrene D. Thus, this study identified δ-cadinene as a novel neutrophil agonist and suggests that δ-cadinene may contribute to the reported immunomodulatory activity of E. purpurea.Item Development of potent isoflavone-based formyl peptide receptor 1 (FPR1) antagonists and their effects in gastric cancer cell models(Elsevier BV, 2023-12) Francavilla, Fabio; Sarcina, Federica; Schepetkin, Igor A.; Kirpotina, Lilya N.; Contino, Marialessandra; Schirizzi, Annalisa; De Leonardis, Giampiero; Khlebnikov, Andrei I.; D'Alessandro, Rosalba; Quinn, Mark T.; Lacivita, Enza; Leopoldo, MarcelloFormyl peptide receptor-1 (FPR1) is a G protein-coupled chemoattractant receptor that plays a crucial role in the trafficking of leukocytes into the sites of bacterial infection and inflammation. Recently, FPR1 was shown to be expressed in different types of tumor cells and could play a significant role in tumor growth and invasiveness. Starting from the previously reported FPR1 antagonist 4, we have designed a new series of 4H-chromen-2-one derivatives that exhibited a substantial increase in FPR1 antagonist potency. Docking studies identified the key interactions for antagonist activity. The most potent compounds in this series (24a and 25b) were selected to study the effects of the pharmacological blockade of FPR1 in NCl–N87 and AGS gastric cancer cells. Both compounds potently inhibited cell growth through a combined effect on cell proliferation and apoptosis and reduced cell migration, while inducing an increase in angiogenesis, thus suggesting that FPR1 could play a dual role as oncogene and onco-suppressor.Item Neuroprotective Effects of Tryptanthrin-6-Oxime in a Rat Model of Transient Focal Cerebral Ischemia(MDPI AG, 2023-07) Plotnikov, Mark B.; Chernysheva, Galina A.; Smol’yakova, Vera I.; Aliev, Oleg I.; Anishchenko, Anna M.; Ulyakhina, Olga A.; Trofimova, Eugene S.; Ligacheva, Anastasia A.; Anfinogenova, Nina D.; Osipenko, Anton N.; Kovrizhina, Anastasia R.; Khlebnikov, Andrei I.; Schepetkin, Igor A.; Drozd, Anastasia G.; Plotnikov, Evgenii V.; Atochin, Dmitriy N.; Quinn, Mark T.The activation of c-Jun N-terminal kinase (JNK) plays an important role in stroke outcomes. Tryptanthrin-6-oxime (TRYP-Ox) is reported to have high affinity for JNK and anti-inflammatory activity and may be of interest as a promising neuroprotective agent. The aim of this study was to investigate the neuroprotective effects of TRYP-Ox in a rat model of transient focal cerebral ischemia (FCI), which involved intraluminal occlusion of the left middle cerebral artery (MCA) for 1 h. Animals in the experimental group were administered intraperitoneal injections of TRYP-Ox 30 min before reperfusion and 23 and 47 h after FCI. Neurological status was assessed 4, 24, and 48 h following FCI onset. Treatment with 5 and 10 mg/kg of TRYP-Ox decreased mean scores of neurological deficits by 35–49 and 46–67% at 24 and 48 h, respectively. At these doses, TRYP-Ox decreased the infarction size by 28–31% at 48 h after FCI. TRYP-Ox (10 mg/kg) reduced the content of interleukin (IL) 1β and tumor necrosis factor (TNF) in the ischemic core area of the MCA region by 33% and 38%, respectively, and attenuated cerebral edema by 11% in the left hemisphere, which was affected by infarction, and by 6% in the right, contralateral hemisphere 24 h after FCI. TRYP-Ox reduced c-Jun phosphorylation in the MCA pool at 1 h after reperfusion. TRYP-Ox was predicted to have high blood–brain barrier permeability using various calculated descriptors and binary classification trees. Indeed, reactive oxidant production was significantly lower in the brain homogenates from rats treated with TRYP-Ox versus that in control animals. Our data suggest that the neuroprotective activity of TRYP-Ox may be due to the ability of this compound to inhibit JNK and exhibit anti-inflammatory and antioxidant activity. Thus, TRYP-Ox may be considered a promising neuroprotective agent that potentially could be used for the development of new treatment strategies in cerebral ischemia.Item Novel Tryptanthrin Derivatives with Selectivity as c–Jun N–Terminal Kinase (JNK) 3 Inhibitors(MDPI AG, 2023-06) Schepetkin, Igor A.; Karpenko, Oleksander S.; Kovrizhina, Anastasia R.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Chekal, Stepan I.; Radudik, Alevtyna V.; Shybinska, Maryna O.; Quinn, Mark T.The c-Jun N-terminal kinase (JNK) family includes three proteins (JNK1-3) that regulate many physiological processes, including cell proliferation and differentiation, cell survival, and inflammation. Because of emerging data suggesting that JNK3 may play an important role in neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease, as well as cancer pathogenesis, we sought to identify JNK inhibitors with increased selectivity for JNK3. A panel of 26 novel tryptanthrin-6-oxime analogs was synthesized and evaluated for JNK1-3 binding (Kd) and inhibition of cellular inflammatory responses. Compounds 4d (8-methoxyindolo[2,1-b]quinazolin-6,12-dione oxime) and 4e (8-phenylindolo[2,1-b]quinazolin-6,12-dione oxime) had high selectivity for JNK3 versus JNK1 and JNK2 and inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 (NF-κB/AP-1) transcriptional activity in THP-1Blue cells and interleukin-6 (IL-6) production by MonoMac-6 monocytic cells in the low micromolar range. Likewise, compounds 4d, 4e, and pan-JNK inhibitor 4h (9-methylindolo[2,1-b]quinazolin-6,12-dione oxime) decreased LPS-induced c-Jun phosphorylation in MonoMac-6 cells, directly confirming JNK inhibition. Molecular modeling suggested modes of binding interaction of these compounds in the JNK3 catalytic site that were in agreement with the experimental data on JNK3 binding. Our results demonstrate the potential for developing anti-inflammatory drugs based on these nitrogen-containing heterocyclic systems with selectivity for JNK3.Item Inhibition of Acetylcholinesterase by Novel Lupinine Derivatives(MDPI AG, 2023-04) Schepetkin, Igor A.; Nurmaganbetov, Zhangeldy S.; Fazylov, Serik D.; Nurkenov, Oralgazy A.; Khlebnikov, Andrei I.; Seilkhanov, Tulegen M.; Kishkentaeva, Anarkul S.; Shults, Elvira E.; Quinn, Mark T.Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment due in part to a severe loss of cholinergic neurons in specific brain areas. AD is the most common type of dementia in the aging population. Although several acetylcholinesterase (AChE) inhibitors are currently available, their performance sometimes yields unexpected results. Thus, research is ongoing to find potentially therapeutic AChE inhibitory agents, both from natural and synthetic sources. Here, we synthesized 13 new lupinine triazole derivatives and evaluated them, along with 50 commercial lupinine-based esters of different carboxylic acids, for AChE inhibitory activity. The triazole derivative 15 [1S,9aR)-1-((4-(4-(benzyloxy)-3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)octahydro-2H-quinolizine)] exhibited the most potent AChE inhibitory activity among all 63 lupinine derivatives, and kinetic analysis demonstrated that compound 15 was a mixed-type AChE inhibitor. Molecular docking studies were performed to visualize interaction between this triazole derivative and AChE. In addition, a structure-activity relationship (SAR) model developed using linear discriminant analysis (LDA) of 11 SwissADME descriptors from the 50 lupinine esters revealed 5 key physicochemical features that allowed us to distinguish active versus non-active compounds. Thus, this SAR model could be applied for design of more potent lupinine ester-based AChE inhibitors.Item Cardioprotective Effects of a Selective c-Jun N-terminal Kinase Inhibitor in a Rat Model of Myocardial Infarction(MDPI AG, 2023-02) Plotnikov, Mark B.; Chernysheva, Galina A.; Smol’yakova, Vera I.; Aliev, Oleg I.; Fomina, Tatyana I.; Sandrikina, Lyubov A.; Sukhodolo, Irina V.; Ivanova, Vera V.; Osipenko, Anton N.; Anfinogenova, Nina D.; Khlebnikov, Andrei I.; Atochin, Dmitriy N.; Schepetkin, Igor A.; Quinn, Mark T.Activation of c-Jun N-terminal kinases (JNKs) is involved in myocardial injury, left ventricular remodeling (LV), and heart failure (HF) after myocardial infarction (MI). The aim of this research was to evaluate the effects of a selective JNK inhibitor, 11H-indeno [1,2-b]quinoxalin-11-one oxime (IQ-1), on myocardial injury and acute myocardial ischemia/reperfusion (I/R) in adult male Wistar rats. Intraperitoneal administration of IQ-1 (25 mg/kg daily for 5 days) resulted in a significant decrease in myocardial infarct size on day 5 after MI. On day 60 after MI, a significant (2.6-fold) decrease in LV scar size, a 2.2-fold decrease in the size of the LV cavity, a 2.9-fold decrease in the area of mature connective tissue, and a 1.7-fold decrease in connective tissue in the interventricular septum were observed compared with the control group. The improved contractile function of the heart resulted in a significant (33%) increase in stroke size, a 40% increase in cardiac output, a 12% increase in LV systolic pressure, a 28% increase in the LV maximum rate of pressure rise, a 45% increase in the LV maximum rate of pressure drop, a 29% increase in the contractility index, a 14% increase in aortic pressure, a 2.7-fold decrease in LV end-diastolic pressure, and a 4.2-fold decrease in LV minimum pressure. We conclude that IQ-1 has cardioprotective activity and reduces the severity of HF after MI.Item Neutrophil Immunomodulatory Activity of Nerolidol, a Major Component of Essential Oils from Populus balsamifera Buds and Propolis(MDPI AG, 2022-12) Schepetkin, Igor A.; Özek, Gulmira; Özek, Temel; Kirpotina, Liliya N.; Kokorina, Polina I.; Khlebnikov, Andrei I.; Quinn, Mark T.Propolis is a resinous mixture of substances collected and processed from various botanical sources by honeybees. Black poplar (Populus balsamifera L.) buds are one of the primary sources of propolis. Despite their reported therapeutic properties, little is known about the innate immunomodulatory activity of essential oils from P. balsamifera and propolis. In the present studies, essential oils were isolated from the buds of P. balsamifera and propolis collected in Montana. The main components of the essential oil from P. balsamifera were E-nerolidol (64.0%), 1,8-cineole (10.8%), benzyl benzoate (3.7%), α-terpinyl acetate (2.7%), α-pinene (1.8%), o-methyl anisol (1.8%), salicylaldehyde (1.8%), and benzyl salicylate (1.6%). Likewise, the essential oil from propolis was enriched with E-nerolidol (14.4%), cabreuva oxide-VI (7.9%), α-bisabolol (7.1%), benzyl benzoate (6.1%), β-eudesmol (3.6%), T-cadinol (3.1%), 2-methyl-3-buten-2-ol (3.1%), α-eudesmol (3.0%), fokienol (2.2%), nerolidol oxide derivative (1.9%), decanal (1.8%), 3-butenyl benzene (1.5%), 1,4-dihydronaphthalene (1.5%), selina-4,11-diene (1.5%), α-cadinol (1.5%), linalool (1.4%), γ-cadinene (1.4%), 2-phenylethyl-2-methyl butyrate (1.4%), 2-methyl-2-butenol (1.3%), octanal (1.1%), benzylacetone (1.1%), and eremoligenol (1.1%). A comparison between P. balsamifera and propolis essential oils demonstrated that 22 compounds were found in both essential oil samples. Both were enriched in E-nerolidol and its derivatives, including cabreuva oxide VI and nerolidol oxides. P. balsamifera and propolis essential oils and pure nerolidol activated Ca2+ influx in human neutrophils. Since these treatments activated neutrophils, the essential oil samples were also evaluated for their ability to down-regulate the neutrophil responses to subsequent agonist activation. Indeed, treatment with P. balsamifera and propolis essential oils inhibited subsequent activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, nerolidol inhibited human neutrophil activation induced by fMLF (IC50 = 4.0 μM) and WKYMVM (IC50 = 3.7 μM). Pretreatment with the essential oils and nerolidol also inhibited human neutrophil chemotaxis induced by fMLF, again suggesting that these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Finally, reverse pharmacophore mapping predicted several potential kinase targets for nerolidol. Thus, our studies have identified nerolidol as a potential anti-inflammatory modulator of human neutrophils.
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