Background
Natural sources hold the promise of discovering new structural classes with distinctive bioactivities for treatment of inflammatory diseases.
Physalis is a tropical plant, which belongs to family Solanaceae [
1].
Physalis plants are well known to be utilized in folk medicine for treating several ailments including malaria, hepatitis, asthma, rheumatism, diabetes and dermatitis also they were used as diuretic, antiseptic, antispasmodic, sedative and analgesic [
2]. Recent medicinal reports showed that
physalis plants exhibited hypoglycemic [
3], anti-inflammatory [
4], antitumor [
5] and antioxidant effects [
6]. There are more than 120 species of the genus
Physalis in the world.
Physalis is known locally in Egypt as Harankash, which is represented by three species:
P. ixocarpa Brat,
P. pubescens L and
P. pruinosa L [
7]. Meanwhile, the scarce reports on
P. pruinosa L. prompted us to study this species.
P. pruinosa was found to possess anti-inflammatory, hypoglycemic, antioxidant and antihyperlipidemic activities [
3,
8].
Scientists are beginning to understand that plants may act as reservoirs for an infinite number of microorganisms, which are known as endophytes [
9]. Endophytes are known to live in a symbiotic relationship interior plant tissue. Moreover, they are able to spend the most of their life cycle inside host plants. It is known that the secondary metabolites isolated from endophytes possess a broad spectrum of pharmacological activities, which include anticancer, antifungal, antiviral, anti-inflammatory and antibacterial activity [
10]. For the past few decades, the discovery rate of active novel chemical entities has been clearly declined. Plant sources are being exhausted for new chemical entities discovery, which are utilized for various therapeutic purposes [
9]. Thus, endophytic microorganisms were found to play an important role in the search for natural bioactive compounds. Only few plants have been studied to date for their endophytic variety and capacity to produce bioactive secondary metabolites. Endophytes are now recognized as exclusive suppliers of bioactive compounds [
9]. It has been discovered that several endophytic fungal strains produce unique compounds with a beneficial effect as anti-inflammatory agents [
11]. In recent studies, it is concluded that endophytic fungi are regarded as a valuable source for biologically active metabolites, which can be used to generate valuable novel anti-inflammatory analogs [
12,
13].
Inflammation including acute and chronic ones can be considered as the body’s response to external stimuli like irritants, pathogens or infections. Chronic inflammation, however, can result from recurrent exposure to potentially hazardous substances or from inadequate control of acute inflammation [
14]. Chronic inflammation-related disorders include cardiovascular, neurological and inflammatory bowel disease, which may develop as a result of persistent inflammatory reactions [
15]. Additionally, chronic inflammation has a role in both physiological and pathological processes of a number of disorders, including Alzheimer’s disease, cancer, gout, obesity, depression and rheumatoid arthritis [
16,
17]. Regulation of the inflammation process is performed
via a precise modulation reaction between the cells and inflammatory mediators. The inflammatory mediators include cyclo-oxygenase and lipoxygenase enzymes, prostaglandin E2, nitric oxide, cytokines (tumor necrosis factor (TNF-
α), interferons (IFN) and interleukins (ILs)) in addition to the transcription factor (NF-
ΚB) [
18]. They are released from the activated inflammatory cells (macrophages, neutrophils and eosinophils). The most widely used traditional treatments for inflammation are currently steroidal and non-steroidal anti-inflammatory drugs, which have analgesic, anti-inflammatory in addition to other curative effects. However, prolonged use of these medications may result in a variety of adverse reactions [
19], including gastrointestinal harm (bleeding, gastric ulcers, etc.) [
20], kidney and liver dysfunction [
20]. However, rational use of the drugs recommends drugs with a higher level of safety. Hence, searching for natural good curative candidates without harmful side effects to replace the conventional anti-inflammatory medications is a pressing issue that must be resolved in the clinical management of inflammation-related diseases [
21].
Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus Physalis as source for anti-inflammatory candidates, the objective of the present study was to isolate for the first time some endophytic fungi from the medicinal plant P. pruinosa. Moreover, comparative evaluation of ex vivo anti-inflammatory activity of the isolated endophytic fungi on LPS-stimulated white blood cells was attempted followed by isolation of the main active metabolites from the most potent anti-inflammatory endophytic fungi. Assessment of ex vivo anti-inflammatory activity of the isolated compounds as well as the mechanisms underlying these effects using computer-aided molecular docking was performed.
Methods
Isolation of endophytic fungi
Healthy leaves of
P. pruinosa L. (
http://www.theplantlist.org/tpl1.1/record/kew-2549665) were collected from a farm owned by the University located in Moderyet El Tahrir District, Beheira Government, Egypt in April 2020. Plant authentication was done by Dr. Mahasen Sedky, Horticultural Research Institute, Cairo, Egypt. A voucher specimen (PP2020) was kept at the Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University. Ethical approval for plant material collection was obtained from the research ethical committee at Faculty of Pharmacy, Alexandria University (PP2020/MN500). Plant collection complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The collected fresh leaves were washed using tape water and then rinsed with distilled water. Furthermore, surface sterilization using 70% ethanol (1 min.) followed by 3.0% sodium hypochlorite (3 min.) was performed. Then rinsing the sterilized samples was done thrice using sterile distilled water (1 min. each). Every sterilized sample was cut into small pieces (approximately 5 mm) and then placed on petri dishes containing potato dextrose agar enriched with ampicillin (50 mg/L), which then incubated at 25 ± 2 °C. For culture purification, subculturing of the tips of the endophytic fungi, which grew out from the leaves was performed using other PDA plates [
22,
23].
Morphological characterization and molecular identification of endophytic isolates
Identification of the endophytic isolates was performed on the basis of macroscopic (fungus culture) and microscopic characters (spore shape and type) [
24] in addition to molecular identification [
25]. DNA extraction was performed as follow. The sample (200 µL) was mixed thoroughly with water (95 µL), solid tissue buffer (95 µL) and proteinase K (10 µL) in a microcentrifuge tube. Then the mixture was incubated at 55ºC for 2 h and centrifuged at 12,000 x g for 1 min. The aqueous supernatant was then transferred to a clean tube (300 µL) and mixed thoroughly with Genomic Binding Buffer (600 µL). In a collection tube, the mixture was transferred to a Zymo-Spin™ IIC-XL column followed by centrifugation at ≥ 12,000 x g for 1 min. In another collection tube, DNA Pre-Wash Buffer (400 µL) was loaded to the column followed by centrifugation at 12.000 xg for 1 min. Adding g-DNA wash buffer (700 µL) was performed followed by centrifugation at 12.000 xg for 1 min. Then 200 µL g-DNA wash buffer was added followed by centrifugation at 12.000 xg for 1 min. Finally, elution buffer (30 µL) was added and then incubated for 5 min. and centrifuged at 12.000 xg for 1 min. Polymerase Chain Reaction (PCR) was utilized for DNA amplification. The PCR was carried out using MyTaq Red Mix mastermix. ITS 1 (with base sequences TCCGTAGGTGAACCTGCGG) and ITS 4 (with base sequences TCCTCCGCTTATTGATATGC) (Invitrogen) were used as primers for DNA amplification. The PCR reaction mixture contained 25 µL MyTaq Red Mix polymerase, 8 µL DNA template, 1 µL forward primer (20 Pico mol), 1µL reverse primer (20 Pico mol) and 15 µL nuclease free water. The mixture was then applied to the thermal cycler (BioRad) using programmed PCR cycle. The amplified DNA was then submitted to a commercial service for sequencing and the base sequence was checked against freely accessible databases like GenBank using the Blast-Algorithmus.
Cultivation and extraction of the fungal strain
For screening, isolation and identification of secondary metabolites from the cultivated fungi, mass growth was carried out using two methods. The first one [
26] (solid rice medium cultivation) was used to cultivate
Stemphylium [
27] species which performed using 500 mL Erlenmeyer flasks containing 80 g rice and 120 mL distilled water (10 flasks), then incubated at 28 °C. Extraction of the fungal cultures was performed by sonication using methanol after three weeks of incubation. The methanolic extracts were then evaporated under reduced pressure. The second method [
22] was used to cultivate
Alternaria [
27] and
Fusarium [
28] species, which was done by inoculation of the endophytic fungi in potato dextrose broth (PDB) medium (12 g potato dextrose per 500 mL distilled water in a 1 L Erlenmeyer flask (10 flasks for each strain) followed by incubation at 28 °C in a shaker incubator (120 rpm) for 2 weeks. The culture was then extracted using ethyl acetate and then sonicated at 40 ° C for 20 min. (three times). Collection of the ethyl acetate layer and then evaporation under vacuum was performed.
The ethyl acetate extract of A. alternata MN615420 (800 mg) cultivated in PDB medium was chromatographed over a normal phase silica gel column (50 g, 2 cm X 90 cm) using hexane: ethyl acetate gradient elution with gradual increase in polarity (0-100%) to yield five fractions (Fractions A–E). Fraction A was purified by normal phase silica gel column chromatography (30 g, 1.5 cm X 80 cm) using a gradient solvent system of methylene chloride: methanol with gradual increase in polarity (10–80%) to afford compound 1 (15 mg), compound 2 (10 mg) and compound 3 (10 mg). Fraction C was subjected to normal phase silica gel column chromatography (30 g, 1.5 cm X 80 cm) using hexane: ethyl acetate gradient elution with gradual increase in polarity (20–90%) followed by sephadex LH-20 column eluted with methanol to obtain 5 mg compound 4. Fraction D was separated by normal phase silica gel column chromatography (20 g, 1.5 cm X 60 cm) using a gradient solvent system of methylene chloride: methanol (20–90%) to yield an inseparable mixture of compounds 5 and 6. Structure elucidation of the isolated compounds was done using NMR and MS techniques.
Alternariol monomethyl ether (1)
Violet crystals, ESIMS negative m/z 271.1 [M – H]−. 1HNMR (500 MHz, DMSO): δH 2.75 (3H, s, 6’-CH3), 3.92 (3 H, s, 5-OCH3), 6.64 (1 H, d, J = 2.1 Hz, H-3’), 6.67 (1 H, d, J = 2.5 Hz, H-4), 6.74 (1 H, d, J = 2.1 Hz, H-5’), 7.25 (1 H, d, J = 2.5 Hz, H-6). 13CNMR (125 MHz, DMSO): δC 25.2 (6’-CH3), 56.2 (5-OCH3), 99.0 (C-2), 99.7 (C-3′), 102.0 (C-4), 103.8 (C-6), 109.1 (C-1’), 118.0 (C-5’), 138.2 (C-1), 138.7 (C-6’), 152.8 (C-2’), 159.0 (C-4’), 164.6 (C-3), 165.3 (C-5), 166.6 (C-7).
3’-Hydroxyalternariol monomethyl ether (2)
Yellow crystals, ESIMS negative m/z 287.2 [M – H]−.1HNMR (500 MHz, DMSO): δH 2.68 (3H, s, 6-CH3), 3.79 (3H, s, 5-OCH3), 6.76 (1H, d, J = 2.0 Hz, H-4), 6.99 (1H, s, H-5), 7.28 (1H, d, J = 2.0 Hz, H-6), 12.51 (1H, s, 3-OH). 13CNMR (125 MHz, DMSO): δC 25.1 (6-CH3), 56.5 (5-OCH3), 100.1 (C-2), 100.3 (C-4), 104.8 (C-6), 111.4 (C-1), 117.5 (C-5), 128.5 (C-6), 131.5 (C-3), 138.4 (C-1), 142.2 (C-2’), 146.5 (C-4), 165.0 (C-3), 165.2 (C-7), 167.1 (C-5).
Alternariol (3)
Colorless crystals, ESIMS negative m/z 257.0 [M – H]−. 1HNMR (500 MHz, DMSO): δH 2.71 (3H, s, 6’-CH3), 6.38 (1 H, d, J = 2.2 Hz, H-3’), 6.65 (1 H, d, J = 2.0 Hz, H-4), 6.72 (1 H, d, J = 2.2 Hz, H-5’), 7.26 (1 H, d, J = 2.0 Hz, H-6), 11.70 (1 H, s, 3-OH). 13CNMR (125 MHz, DMSO): δC 25.7 (6’-CH3), 97.8 (C-2), 101.3 (C-4), 102.1 (C-3′), 104.8 (C-6), 109.4 (C-1′), 118.0 (C-5′), 138.5 (C-1), 138.8 (C-6’), 153.1 (C-2′), 158.8 (C-4′), 164.5 (C-3), 165.1 (C-7), 165.9 (C-5).
A-Acetylorcinol (4)
Yellow crystals, ESIMS positive m/z 167.3 [M + H]+. 1HNMR (500 MHz, DMSO): δH 2.07 (3 H, s, H-9), 3.45 (2 H, s, H-7), 6.04 (2 H, m, H-2, H-6), 6.09 (1 H, m, H-4). 13CNMR (125 MHz, DMSO): δC 29.6 (C-9), 50.5 (C-7), 101.2 (C-4), 108.2 (C-2, C-6), 136.9 (C-1), 158.8 (C-3, C-5), 206.6 (C-8).
Mixture of tenuazonic acid (5) and allo-tenuazonic acid (6)
Viscous, yellow oil, ESIMS negative m/z 196.1 [M – H]−.1HNMR (500 MHz, CD3OD): δH 0.81(m, H-10), 0.82 (m, H-10), 1.16 (m, H-11), 1.35–1.42 (m, H-9), 1.90 (m, H-8), 2.35 (s, H-7), 3.61 (s, H-5), 3.78 (s, H-5). 13CNMR (125 MHz, CD3OD): δC 11.6 (C-10), 15.2 (C-11), 22.8 (C-9), 25.6 (C-7), 36.4 (C-8), 36.8 (C-8), 63.7 (C-5), 65.5 (C-5), 102.5 (C-3), 177.5 (C-2), 177.8 (C-2), 193.6 (C-6), 193.7 (C-6).
Evaluation of cytotoxicity and anti-inflammatory activity of the tested extracts and isolated compounds
Human white blood cells isolation and cultivation
Collection of a whole blood specimen was performed using a sterile heparin tube, then an aliquot (1 mL) of blood was transferred into a centrifuge tube (15 mL), which filled to capacity with fresh cold lysing solution. The tube was then inverted at room temperature for ~ 10 min. till the liquid turned into clear red. Centrifugation of specimens was done at 2000 rpm (4
oC) for 10 min., then decantation of supernatant was performed and the tubes were permitted to drain. In 10 mL cold phosphate buffer saline (137 mM NaCl, 10 mM Na
2HPO
4, 2.7 mM KCl and 10 mM KH
2PO
4, pH 7.4), the pellets (WBCs) were suspended Followed by recentrifugation. The pellets were then resuspended using RPMI culture medium, which contained 2% L-glutamine and 10% fetal bovine. Then, dye exclusion method for assessment of WBCs viability and counting was utilized. Mixing of 50 µL cell suspension with the same volume of trypan blue staining solution (0.5%) was performed followed by hemocytometer loading. In each of the four corner quadrants (A, B, C, D), viable and nonviable cells were counted [
29].
Calculations
N / ml = Mean of WBCs counting x10
4
× D
N: Number of viable or nonviable cells.
D: Sample dilution (1:1 with the trypan blue).
$$\% \,Cell\,viability = \frac{{Number\,of\,viable\,\,cells}}{{Total\,number\,of\,cells}} \times 100$$
To use the cells for assays, at least 90% of them must be viable after incubating the culture in CO2 incubator for six days. Seeding of WBCs was performed (100,000 cells/ well) in a 96 well cell culture plate followed by incubation in CO2 incubator (5% CO2 and 90% relative humidity at 37 °C).
All experiments were performed in accordance with relevant guidelines and regulations.
Evaluation of cytotoxicity of the studied extracts and isolated compounds relative to piroxicam (MTT assay)
200 µL of cultured medium (containing 100,000 WBCs / well (96-well cell culture plate)) were seeded with variable concentrations of the studied extracts/ isolated compounds (0, 3.125, 6.25, 12.5, 25 and 50 µg/mL) in RPMI medium without both fetal bovine serum and piroxicam (the standard anti-inflammatory drug) then incubation was performed in CO
2 incubator for 72 h (37 °C, 5% CO
2 and 90% relative humidity). Then MTT solution was added to each well after 72 h of incubation. 20 µL volume of plates were incubated in CO
2 incubator for 3 h to enable the MTT to be reacted. Following incubation, the plates were centrifuged for 10 min. at 1650 rpm, then the medium was discarded. MTT byproduct, formazan crystals, was re-suspended using DMSO (100 µL) and then measurement of readings was performed using optima spectrophotometer at wavelength 570 nm for safe dose determination that cause 100% cell viability [
30].
The % viability was calculated as follow: (AT-Ab /AC-Ab) x 100
AT = Mean absorbances of cells treated with different concentration of each studied extract or isolated compound.
AC = Mean absorbances of the control untreated cells (only culture medium).
Ab = Mean absorbances of cells treated with vehicle of the studied extracts or isolated compounds (RPMI without fetal bovine serum).
Using the percent viability derived from each studied extract/ isolated compound serial dilutions, the cytotoxicity assay of the drug was expressed as EC100 that calculated using the Graphpad Instat software (GraphPad Software Inc, California).
Determination of the effective anti-inflammatory concentrations (EAICs) of the used treatments in lipopolysaccharides (LPS)-stimulated human WBC’s culture
50 µL culture medium, which contained 100,000 of human WBCs, was dispensed per well in a 96 well plate. Inflammation induction was performed by adding 50 µL of LPS (20 µg/mL) to the plated cells followed by incubation in CO
2 incubator. After 24 h, centrifugation of the plate (1650 rpm) was done for 5 min., afterward the supernatants were discarded. After that, 200 µL of crude extracts, isolated compounds or piroxicam were added using serial concentrations (0, 3.125, 6.25, 12.5, 25 and 50 µg/ml in culture media). Only cell culture medium was present in the control cells. Plates incubation occurred for extra 72 h in CO
2 incubator. Following incubation, cell proliferations were assessed using MTT (as previously mentioned). Cell proliferations assessment was done using stimulation index (SI) [
31].
Stimulation index = (mean absorbance of LPS-stimulated cells or LPS-stimulated cells treated with different concentrations of the studied extracts or isolated compounds / absorbance of control untreated cells).
Instate graph pad was used to determine the effective anti-inflammatory concentrations (EAICs) of the studied extracts and isolated compounds that could restore the aberrant proliferation of LPS-stimulated cells to the normal proliferation of control, untreated cells (SI = 1).
Suspension of cell pellets was then made in solution R1 (50 µL), then it was mixed thoroughly for 30 s and incubated at room temperature (1 min.). 300 µL solution R2 was added, mixed thoroughly for 30 s and then centrifuged for 3–5 min. at 4ºC. In a spin column, the supernatant was transferred followed by centrifugation for 30 s at 14,000 rpm (4ºC). After removing the flow-through, working wash buffer (300 µL) was added into the spin column followed by centrifugation for 30 s (repeated twice). Recentrifugation of the spin column was performed at 10,000 rpm (1 min.) and then transferred to a sterile micro centrifuge tube (1.5 mL). Elution buffer (30 µL) was added to the central of the membrane, then incubated at room temperature (1 min.) and then centrifuged for 30 s at 14,000 rpm at 4ºC. Determination of the optical density (OD) of the extracted RNA was performed through measuring the absorbance (A260 nm) and purity (A280 nm) using spectrophotometer then kept in -80 °C till real time PCR [
32].
Synthesis of cDNA from RNA extracted from untreated and treated LPS-stimulated human white blood cells
Total RNA or nuclease-free water (2 µg) in addition to oligo dT primer (1 µL) were mixed gently with nuclease-free water in PCR tubes in a total volume of 12 µL, centrifuged, incubated at 65ºC (5 min.) using PCR machine and then put back on ice instantly. 5X reaction buffer (4 µL), dNTPs mix (2 µL), RNase inhibitor (1 µL) and reverse transcriptase (1 µL) or nuclease-free water (1 µL) in place of reverse transcriptase for reverse transcriptase negative control were mixed gently with prior mixture, spin down and then incubated at 42ºC (60 min.) followed by heat inactivation at 70ºC (5 min.) in PCR machine [
32].
Determination of IL-1β, TNF-α, INF-γ and GADPH expression level by real time polymerase chain reaction (PCR)
In PCR tubes, a volume of 12.5 µL of 2 X SYBR green master mix was mixed with 5µL of cDNA 0.5 µL of 10 pico moles/mL forward primer and 0.5 µL of 10 pico moles/mL reverse primer for each primer. As for the reference tube, 0.5 µL of 10 pico moles/mL forward primer of
β-actin and 0.5 µL of 10 picomoles/mL for reverse primer of
β-actin were added. The other tube was used as a non-template control (NTC), to assess for reagent contamination or primer dimers by adding 1 µL of nuclease-free water instead of template used. The tubes were gently mixed with 6.5 µL nuclease free water without creating bubbles (bubbles will interfere with the fluorescence detection) and spinned for few seconds. Samples were placed in the cycler and start the program as follows; 1 cycle of 95ºC for 10 min. (initial denaturation), followed by 40 cycles of 95ºC for 15 s (denaturation), 60ºC for 30 s (annealing) and 72ºC for 30 s (extension) [
32].
Calculations
Expressions fold levels of gene calculated by:
ΔCt normal = Ct normal untreated cells – Ct reference.
ΔCt tested sample = Ct tested sample-treated cells – Ct reference.
ΔCt induced =- Ct LPS-exposed cells – Ct reference.
In case of genes:
ΔΔCT tested sample = ΔCt tested sample– ΔCt normal.
ΔΔCT induced = ΔCt induced – ΔCt normal.
In case of GAPDH:
ΔΔCTtested plant extract = ΔCt normal – ΔCt tested plant extract.
ΔΔCTinduced = ΔCt normal – ΔCt induced.
Fold change in gene expression = log (2
−ΔΔCT
)
Where:
Ct tested sample: Threshold cycle value of genes of extracted mRNA of the studied extracts/isolated compounds treated-LPS-stimulated WBCs which is the cycle number at which fluorescence produced during a reaction crosses the fluorescence threshold.
Ct reference: Threshold cycle value of GAPDH which is utilized in normalization.
Ct normal: Threshold cycle value of genes of extracted mRNA of untreated control WBCs.
Ct induced: Threshold cycle value of gene of extracted mRNA of LPS-stimulated WBCs.
Primers
TNF-α | F, CTCTTCTGCCTGCTGCACTTTG |
R, ATGGGCTACAGGCTTGTCACTC |
IL-1β | F, CCACAGACCTTCCAGGAGAATG |
R, GTGCAGTTCAGTGATCGTACAGG |
INF-γ | F, GAGTGTGGAGACCATCAAGGAAG |
R, TGCTTTGCGTTGGACATTCAAGTC |
GAPDH | F, GGATTTGGTCGTATTGGG |
R, GGAAGATGGTGATGGGATT |
In silico docking studies
The Schrödinger Maestro 11.8 package (LLC, New York, NY) was employed in order to predict the binding mode of the most potent bioactive compounds [
33].
Ligand structures preparation
The two-dimensional structures of the isolated compounds along with the reference drug; piroxicam (CID 54,676,228) were obtained from PubChem database (
https://pubchem.ncbi.nlm.nih.gov/) of the National Centre for Biotechnology Information in sdf file format. ChemDraw software (Cambridge So Corporation, Cambridge, USA) was used to draw the structures that weren’t included in PubChem database after searching through various literature. The chemical structure of each substance was imported into the Maestro 11.8 panel interface (Maestro, version 11.8, 2018, Schrodinger, USA) once the dataset was generated. The 3D structure was created using the LigPrep 2.3 module (LigPrep, version 2.3, 2018, Schrodinger, USA), which was also used to search for various conformers. To geometrically optimize each ligand structure and compute partial atomic charges, the OPLS (OPLS 2005, Schrodinger, USA) force field was used.
Target protein preparation
The crystal structures of human IL-1
β (PDB ID 4DEP) [
34], TNF-
α (PDB ID 2AZ5) [
35] and INF-
γ (PDB ID 6E3K) [
36] were obtained from the RCSB Protein Data Bank (PDB). Using the protein preparation module of Schrodinger’s Maestro Molecular Modelling Suit (Schrödinger Release 2015-2), the protein crystallographic structure was loaded and created.
Grid box generation
A grid box with dimensions of 15 × 15 × 15 oA around the centroid of the co-crystallized ligand and a grid spacing of approximately 0.375 oA was used to define the active site for docking on the prepared X-ray crystal structure of the targeted proteins. The targeted proteins crystal structure was subjected to binding interaction examination using this grid box size with the isolated compounds along with the reference drug.
Molecular docking
The Glide 11.8 module (Glide, version 11.8, 2018, Schodinger, USA) was used to perform flexible docking on the minimized and refined compounds while utilizing the Glide default parameters. The Glide-Dock program’s empirical scoring functions was used to generate modeling scores. In order to investigate the most favorable binding modes of ligands, the Maestro interface, which displayed the 2D and 3D ligand-target protein interactions (including hydrogen bonds, ion-pair and hydrophobic interactions) was employed.
Docking process validation
A pose selection approach was used to re-dock piroxicam ligand into the various binding sites of 4DEP, 2AZ5 and 6E3K in order to successfully validate the docking protocol. This was followed by estimating the root mean square deviation (RMSD) of the anticipated pose to the co-crystallized one piroxicam. The results showed high docking accuracy because they were less than 1 oA (a preselected cut-off/threshold value). The re-docked complexes were superimposed onto the reference co-crystallized complexes with low root mean square deviation (RMSD) values (0.52 Å for 2AZ5, 0.59 for 6E3K and 0.65 for 4DEP).
Statistical analysis
Each experiment was carried out in triplicate. All biological data were statistically analyzed using one-way analysis of variance (ANOVA), with p values less than 0.05 being regarded as significant. A neighbour-joining phylogenetic trees were constructed using MEGA X. Graphpad Instat (GraphPad Software Inc, California) was used to calculate EC100 values and construct the dose response curves.
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