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We examined the thymoquinone induced inhibition of purified F1 or membrane bound F1FO
E. coli ATP synthase. Both purified F1 and membrane bound F1FO were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F1Fo and purified F1 preparations. Moreov...
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... inhibition of null strain (pUC118/DK8) by TQ was observed as this strain lacks ATP synthase. Limiting glucose assay contained 3mM glucose and OD 595 was measured till no fur- ther growth occurred which takes about 20 hr time. Growth on succinate plate may take up to 72 hr. There is growing interest in the use of natural compounds as antimicrobial and antitumor agents individually or in combination with other such molecules [21,44,45]. Several phytochemicals have been shown to have dietary benefits and are potential anti-tumor or antimicrobial agents [28,31,46]. For centuries TQ has been used as a natural therapeutic product [47,48]. The goal of this study was to determine if the antimicrobial or anticancer properties of TQ may be associated with the inhibition of ATP synthase. Therefore, we examined TQ effects on ATPase activity and on growth inhibition profiles of E . coli to examine the potential of ATP synthase as a molecular target. TQ fully inhibited both purified F 1 and membrane bound F 1 F o ATP synthase with IC 50 ~36.95 μ M (Fig 3). This is in agreement with multiple previous studies where it was shown that the inhibitory profiles of both F 1 F o membrane preparations as well as isolated purified F 1 are the same [24,49,50,51,52,53]. It is interesting to note that in a previous study simple phenolic compounds, dihydrothymoquinone, hydroquinone, resorcinol, or catechol, structurally related to TQ, resulted in partial or incomplete inhibition of ATP synthase [54]. Resveratrol, piceatannol, and quercetin inhibited ATP synthase X-ray crystal structures show that the polyphenol binding pocket for resveratrol, piceatannol, and quercetin is contributed by residues from α , β , and γ -subunits [19]. Moreover, several polyphenolic compounds structurally related to TQ (Fig 1) were previously shown to bind to the polyphenol binding pocket [24,34,54] identified by Walker and colleagues [19]. Therefore, there is a high possibility that the-CH 3 group of TQ forms hydrophobic non-polar interactions with γ Gln274, γ Thr-277, β Ala-264, β Val-265, γ Ala- 270, γ Thr-273, γ Glu-278, α Gly-282, or α Glu-284 residues. TQ bound X-ray structure of ATP synthase and or mutagenic analysis of above residue should be able to confirm the involvement of above residues in TQ binding. TQ induced inhibition was also found to be completely reversible. Passage through centrifuge columns dissociates TQ from the inhibited F 1 and resulted in restored enzyme function. Dilution of purified F 1 or membrane lowers inhibitor concentration and allowed recovery of ATPase activity. These results indicate that the interaction between inhibitor and the enzyme is non-covalent, as has been observed in previous studies examining the inhibition of ATP synthase by several polyphenolic molecules ...
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... ectopic ATP synthase may be a suitable molecular target for inhibiting HIV-1 proliferation in vivo [14]. A variety of natural and modified plant based molecules are known to induce either complete or partial inhibition of ATP synthase with potential resulting health benefits [7,15,16,17,18]. Some health benefits of fruits, vegetables, and other phytochemicals are credited to the polyphenols present in them. These phytochemicals are known for their antioxi- dants, chemopreventive, chemotherapeutic, and anti-microbial properties [7,19,20,21,22,23]. Some dietary polyphenolic compounds were shown to block the action of cell constituents that promote growth of tumor cells by binding to the multiple molecular targets in the body, including ATP synthase [19,24]. Thymoquinone (TQ) is a major phytochemical compound found in the medicinal plant Nigella sativa an annual flowering plant in the family Ranunculaceae (Fig 1). Thymoquinone has been tested against many cancer cell lines and has exhibited potent inhibitory effects on lung, prostate, and breast cancer studies [25,26,27]. It is also known to have anti-oxidant, anti-inflammatory and anti-diabetic, antibacterial, antifungal, antitussive, and neuroprotective effects [28,29,30,31,32]. Although TQ is being used for centuries and has been observed to be effective against many disease conditions but its mode of action or molecular target is not known. Previ- ous studies suggested that the dietary benefits of several polyphenolic compounds could be associated with their interaction with ATP synthase. For this purpose, we studied the inhibitory effects of thymoquinone on F 1 F o ATP synthase and the growth of E . coli cells. Our results show that thymoquinone strongly inhibits ATPase activity and bacterial growth, thereby suggesting that the beneficial effect of thymoquinone as antitumor or antimicrobial agent may in part be linked to its inhibition of ATP synthase. Thymoquinone with 99% purity (274666-5G) was purchased from Sigma-Aldrich Chemical Company. TQ is unstable in aqueous solution and is light sensitive therefore it was dissolved in ...
Citations
... Phytopolyphenols are a group of natural products found in plants including fruits and vegetables. 85) Our group and others have shown that E. coli F-ATPase is inhibited by phytopolyphenols, such as piceatannol, resveratrol, quercetin, curcumin, thymoquinone, safranal, and olive phenolics 20,32,[86][87][88] (Table 1). As described above, the interface between the β subunit and the C-terminal regions of the γ subunit is important for triggering rotation of the γ subunit, 23) and piceatannol, resveratrol, and quercetin bind to this interface. ...
Proton pumping ATPases, both F-type and V/A-type ATPases, generate ATP using electrochemical energy or pump protons/sodium ions by hydrolyzing ATP. The enzymatic reaction and proton transport are coupled through subunit rotation, and this unique rotational mechanism (rotational catalysis) has been intensively studied. Single-molecule and thermodynamic analyses have revealed the detailed rotational mechanism, including the catalytically inhibited state and the roles of subunit interactions. In mammals, F- and V-ATPases are involved in ATP synthesis and organelle acidification, respectively. Most bacteria, including anaerobes, have F- and/or A-ATPases in the inner membrane. However, these ATPases are not believed to be essential in anaerobic bacteria since anaerobes generate sufficient ATP without oxidative phosphorylation. Recent studies suggest that F- and A-ATPases perform indispensable functions beyond ATP synthesis in oral pathogenic anaerobes; F-ATPase is involved in acid tolerance in Streptococcus mutans, and A-ATPase mediates nutrient import in Porphyromonas gingivalis. Consistently, inhibitors of oral bacterial F- and A-ATPases, such as phytopolyphenols and bedaquiline, strongly diminish growth and survival. Herein, we discuss rotational catalysis of bacterial F- and A-ATPases, and discuss their physiological roles, focusing on oral bacteria. We also review the effects of ATPase inhibitors on the growth and survival of oral pathogenic bacteria. The features of the catalytic mechanism and unique physiological roles in oral bacteria highlight the potential for proton pumping ATPases to serve as targets for oral antimicrobial agents.
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... However, this does not explain the discrepancy in reducing cell density at lower TQ concentrations (Figure 3) when ROS levels were also low ( Figure 4). These differences may be due to a different mechanism described by Ahmad et al. [26]. They showed that lower concentrations of TQ (<150 µM) strongly reduced the growth of E. coli cells by inhibiting the activity of membrane ATPase but did not kill them. ...
The aim of the present study was to evaluate the potential protective effect of glutathione (GSH) on Escherichia coli cells grown in a high concentration of thymoquinone (TQ). This quinone, as the main active compound of Nigella sativa seed oil, exhibits a wide range of biological activities. At low concentrations, it acts as an antioxidant, and at high concentrations, an antimicrobial agent. Therefore, any interactions between thymoquinone and glutathione are crucial for cellular defense against oxidative stress. In this study, we found that GSH can conjugate with thymoquinone and its derivatives in vitro, and only fivefold excess of GSH was sufficient to completely deplete TQ and its derivatives. We also carried out studies on cultures of GSH-deficient Escherichia coli strains grown on a minimal medium in the presence of different concentrations of TQ. The strains harboring mutations in gene ΔgshA and ΔgshB were about two- and fourfold more sensitive (256 and 128 µg/mL, respectively) than the wild type. It was also revealed that TQ concentration has an influence on reactive oxygen species (ROS) production in E. coli strains—at the same thymoquinone concentration, the level of ROS was higher in GSH-deficient E. coli strains than in wild type.
... It was estimated that thymoquinone might prevent the synthesis of DNA and or block the ATP synthase enzyme synthesis of microorganisms. 36 In the past, researchers have shown that thymoquinone induced apoptosis in leukemia cells by the mechanism of loss of mitochondrial membrane potential. 37 In a recent study, thymoquinone exhibited strong leishmanicidal efficacy against promastigotes and amastigotes of L. infantum and L. tropica. ...
... The antileishmanial potential of thymoquinone on the promastigote stage of L. donovani was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide [MTT] cell viability assay using the method described earlier. 36 Briefly, promastigotes at a cell density of 2 × 10 6 cells/mL (100 μL) were harvested from their exponential growth phase and incubated in a BOD incubator at 24°C for 72 h in a 96-well tissue culture plate. Then, 100 μL of thymoquinone was added in two-fold serial dilutions, from 100 μM (100, 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390, 0.195, and 0.1 μM). ...
Visceral leishmaniasis (VL) or kala-azar is a vector-borne dreaded protozoal infection that is caused by the parasite Leishmania donovani. With increases in the dramatic infection rates, present drug toxicity, resistance, and the absence of an approved vaccine, the development of new antileishmanial compounds from plant sources remains the keystone for the control of visceral leishmaniasis. In this study, we evaluated the leishmanicidal effect of thymoquinone against L. donovani with an in vitro and ex vivo model. Thymoquinone exhibited potent antipromastigote activity with IC50 and IC90 concentrations achieved at 6.33 ± 1.21 and 20.71 ± 2.15 μM, respectively, whereas the IC50 and IC90 concentrations were found to be 7.83 ± 1.65 and 27.25 ± 2.20 μM against the intramacrophagic form of amastigotes, respectively. Morphological changes in promastigotes and growth reversibility study following treatment confirmed the leishmanicidal effect of thymoquinone. Further, thymoquinone exhibited leishmanicidal activities against L. donovani promastigote through cytoplasmic shrinkage, membrane blebbing, chromatin condensation, cellular and nuclear shrinkage, and DNA fragmentation, as observed under scanning and transmission electron microscopy analyses. The antileishmanial activity was exerted via programmed cell death as proved by exposure of phosphatidylserine, DNA nicking by TUNEL assay, and loss of mitochondrial membrane potential. Thymoquinone at a concentration of 200 μM was devoid of any cytotoxic effects against mammalian macrophage cells. Thymoquinone showed strong leishmanicidal activity against L. donovani, which is mediated via an apoptosis mode of parasitic cell death, and accordingly, thymoquinone may be the source of a new lead molecule for the cure of VL.
... In addition, TQ induced inhibition of ATP-synthaseexpressing wild-type E. coli cell growth, and the non-inhibition of ATPase gene-deleted null control cells indicates that ATP synthase is a molecular target for TQ. 298 TQ also acted against methicillin-resistant S. aureus (MRSA) strains with MIC values in the range of 8-16 µg mL −1 and an MIC 90 of 16 µg mL −1 , and showed bactericidal and longer post-antibiotic effects and low inclination against MRSA. Transmission electron microscopy of TQ-treated MRSA indicated damage to cell walls and cell membranes, suggesting a novel class of anti-bacterial agents. ...
The dietary phytochemical thymoquinone (TQ), belonging to the family of quinones, mainly obtained from the black and angular seeds of Nigella sativa, is one of the promising monoterpenoid hydrocarbons, which has been receiving massive attention for its therapeutic potential and pharmacological properties. It plays an important role as a chemopreventive and therapeutic agent in the treatment of various diseases and illnesses. The aim of this review is to present a summary of the most recent literature pertaining to the use of TQ for the prevention and treatment of various diseases along with possible mechanisms of action, and the potential use of this natural product as a complementary or alternative medicine. Research findings indicated that TQ exhibits numerous pharmacological activities including antioxidant, anti-inflammatory, cardioprotective, hepatoprotective, antidiabetic, neuroprotective, and anticancer, among others. Conclusions of this review on the therapeutic aspects of TQ highlight the medicinal and folk values of this compound against various diseases and ailments. In short, TQ could be a novel drug in clinical trials, as we hope.
... Respiration was found to be significantly enriched in periodontally healthy cats, and V-Type ATP synthase was significantly more abundant among the diseased groups. ATP synthase, a highly conserved enzyme, is the principal means of energy production in microorganisms 34,35 . A higher abundance of a molecule involved with energy production in the diseased groups was not surprising considering the high bacterial load and microbial biofilm formation in periodontal disease. ...
... The production of ATP by bacteria can occur by substrate-level phosphorylation of fermentable carbon sources or by oxidative phosphorylation through the respiratory chain and ATP synthase 37 . ATP synthase 37 and studies have revealed that ATP synthase is an appropriate target for antimicrobials 34,35,37 . Moreover, Schulz et al. 38 characterized a type of Na + -driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum, which is also noticeably present in dental plaque biofilms. ...
The subgingival microbial communities of domestic cats remain incompletely characterized and it is unknown whether their functional profiles are associated with disease. In this study, we used a shotgun metagenomic approach to explore the functional potential of subgingival microbial communities in client-owned cats, comparing findings between periodontally healthy cats and cats with naturally occurring chronic periodontitis, aggressive periodontitis, and feline chronic gingivostomatitis. Subgingival samples were subjected to shotgun sequencing and the metagenomic datasets were analyzed using the MG-RAST metagenomic analysis server and STAMP v2.1.3 (Statistical Analysis of Metagenomic Profiles) software. The microbial composition was also described to better understand the predicted features of the communities. The Respiration category in the level 1 Subsystems database varied significantly among groups. In this category, the abundance of V-Type ATP-synthase and Biogenesis of cytochrome c oxidases were significantly enriched in the diseased and in the healthy groups, respectively. Both features have been previously described in periodontal studies in people and are in consonance with the microbial composition of feline subgingival sites. In addition, the narH (nitrate reductase) gene frequency, identified using the KEGG Orthology database, was significantly increased in the healthy group. The results of this study provide preliminary functional insights of the microbial communities associated with periodontitis in domestic cats and suggest that the ATP-synthase and nitrate-nitrite-NO pathways may represent appropriate targets for the treatment of this common disease.
... The membrane-bound ATPase activity of E. coli was also inhibited by eugenol (185) or carvacrol (183) [174]. Similar results were obtained for thymoquinone that completely inhibited both purified F 1 and membrane-bound F 1 F 0 E. coli ATP synthase, and the process of inhibition was fully reversible [175]. ...
This review presents the comprehensive knowledge about the bidirectional relationship between polyphenols and the gut microbiome. The first part is related to polyphenols’ impacts on various microorganisms, especially bacteria, and their influence on intestinal pathogens. The research data on the mechanisms of polyphenol action were collected together and organized. The impact of various polyphenols groups on intestinal bacteria both on the whole “microbiota” and on particular species, including probiotics, are presented. Moreover, the impact of polyphenols present in food (bound to the matrix) was compared with the purified polyphenols (such as in dietary supplements) as well as polyphenols in the form of derivatives (such as glycosides) with those in the form of aglycones. The second part of the paper discusses in detail the mechanisms (pathways) and the role of bacterial biotransformation of the most important groups of polyphenols, including the production of bioactive metabolites with a significant impact on the human organism (both positive and negative).
... It has been reported that thymoquinone possesses hepatoprotective, antidiabetic, antifungal, anticancer, and neuroprotective properties [10,14,15]. It is well established that the functional mechanism of thymoquinone is to alter the biochemical reactions associated with the reactive oxygen species (ROS) generation [10]. ...
This study reports the observed synergy in antimicrobial and anticancer activity of thymoquinone and piperine, encapsulated in porous guar gum micro-vehicle. Natural therapeutics like piperine and thymoquinone showed less effectivity in human medical trials due to their hydrophobicity leading to poor clinical efficacy. To overcome this problem, we have developed a delivery strategy by using guar gum, a natural biodegradable biopolymer. The successful encapsulation of phytochemicals and the microstructures of gum micro-vehicles were confirmed by Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), Field emission scanning electron microscopy (FESEM) and UV-Vis spectra analysis. We also report here a significant decrease in minimum inhibitory concentration (MIC) value and synergistic bactericidal activity against four different bacterial strains and observed remarkably low median lethal dose (LD50) value against human hepatocellular carcinoma cell lines along with pH-responsive delivery of therapeutic in the case of combinatorial therapy. In our overall study, we analyze and discuss the structure, efficacy, and delivery of our designed natural therapeutic amalgamation to pave the way for augmenting the use of phytochemicals in medical applications.
... The 6-helices or a 5-helices structure is used as a receptor for protein or peptide docking [39]. Scientists are also looking at molecules that could block furin, which could be investigated as possible therapies [41,[49][50][51]. ...
SARS-CoV-2 has a positive sense RNA genome of 29.9 kb in size, showing high sequence similarity to the BAT-CoV, SARS-CoV, MERS-CoV. SARS-CoV-2 is composed of 14 open reading frames (ORFs), which encodes for a total of 27 proteins divided into structural and non-structural proteins (NSPs). The fundamental structural protein-encoding genes are a spike protein (S) gene, envelope protein (E) gene, a membrane protein (M) gene, and a nucleocapsid protein (N) gene. They make about 33% of the entire genome and are vital for the viral life cycle. Rest 67% is distributed among different NSPs (such as Mpro, helicase, and RNA-dependent RNA polymerase) encoding genes across the ORFs, which are involved in virus-cell receptor interactions during viral entry. Researchers are trying to formulate vaccines, therapeutic antibodies or protein-targeted antiviral drugs to control the spread. This review proceeds stepwise through the COVID-19 outbreak, structural and genomic organization, entry mechanism, pathogenesis, and finally highlighting the essential proteins involved at each step that might be potential targets for drug discovery. Currently, approved treatment modalities consist of only supportive care and oxygen supplementation. This review is established on the current knowledge that has expanded on structural motifs and topology of proteins and their functions.
... In detail, for carvacrol and thymol, whose anti-Campylobacter activity was also studied by Alphen et al. [26], Kelly et al. [27], Upadhyay [28] and others, our data confirmed the published data, in terms of good antimicrobial activity against C. jejuni. On the other side, when thymoquinone was studied for its activity against S. aureus and P. aeruginosa, no effects were observed, although it was reported to be effective against Vibrio parahaemolyticus [29] and E. coli [30]. In the present study, thymoquinone showed antimicrobial activity against C. jejuni that was comparable to carvacrol and thymol, which also confirms the better antimicrobial activity of thymoquinone against Gram-negative bacteria. ...
The culinary herb Satureja montana, known as winter savory, is an ingredient of traditional dishes known in different parts of the world. As an ingredient of foods it has the potential to improve their safety. In this study, the herb’s activity was investigated against Campylobacter jejuni, the leading cause of the most prevalent bacterial gastroenteritis worldwide. The ethanolic extract and essential oil of the herb were chemically characterized and six pure compounds—carvacrol, thymol, thymoquinone, p-cymene, γ-terpinene, and rosmarinic acid—were chosen for further analysis. The antimicrobial activity of the ethanolic extract (MIC 250 mg/L) was 4-fold higher compared to the essential oil. Carvacrol, thymol and thymoquinone had the strongest antimicrobial effect (MIC 31.25 mg/L) and a strong synergistic activity between carvacrol and thymol was determined (FICi 0.2). Strong inhibitory effect on C. jejuni efflux pumps (2-fold inhibition) and disruption of membrane integrity (> 80% disruption) of the herb were determined as modes of action. For resistance against the herb, C. jejuni need efflux pumps, although increased resistance against this herb does not co-occur with increased efflux pump activity, as for antibiotics. This study shows the potential of a common culinary herb for the reduction of the food pathogen C. jejuni without increasing resistance.
... Nigella sativa (Black seed) contain main bioactive active constituent of Thymoquinone (THQ) i.e. Ranunculaceae family, grows commonly in Eastern Europe, Middle East, Western Asia, and Mediterranean countries [10][11][12][13][14]. Nigella sativa seeds are traditionally used for the treatment of fever, cough, bronchitis, asthma, influenza, rheumatism, and headache in the Middle East regions, India, Pakistan, and Northern Africa [15][16][17]. ...
Abstract To formulate a nanoformulation (PLGA-NPs) and to improve brain bioavailability for thymoquinone (THQ) through intranasal (i.n.) drug delivery, using a newly UHPLC-PDA developed the method and validated. Five different THQ-PLGA-NPs (THQ-N1 to THQ-N5) were prepared by emulsion solvent evaporation method. A new UHPLC method developed and validated for biodistribution studies in the rat’s brain, lungs and plasma. Optimized-THQ-N1-NPs showed a particle size of 97.36 ± 2.01 nm with a low PDI value of 0.263 ± 0.004, ZP of − 17.98 ± 1.09, EE of 82.49 ± 2.38% and DL of 5.09 ± 0.13%. THQ-N1-NPs showed sustained release pattern via in vitro release profile. A bioanalytical method was developed by UHPLC-PDA and validated for the evaluation of pharmacokinetics parameters, biodistribution studies, brain drug-targeting potential (89.89 ± 9.38%), and brain-targeting efficiency (8075.00 ± 113.05%) studies through intranasal administration which showed an improved THQ-brain- bioavailability, compared to i.v. Moreover, THQ-PLGA-NPs improved the seizure threshold treatment i.e. epilepsy increasing current electroshock (ICES) rodent models induced seizures in rats. A significant role of THQ-PLGA-NPs with high brain targeting efficiency of the nanoformulations was established. The reported data supports the treatment of epilepsy.
