Biological Evaluation and Computational Studies of Methoxy- avones from Newly Isolated Radioresistant Micromonospora Aurantiaca Strain TMC-15 Wasim Sajjad National University of Medical Sciences Mahnoor Nadeem Quaid-i-Azam University Faculty of Biological Sciences Tayyaba Alam Quaid-i-Azam University Faculty of Biological Sciences Asim ur Rehman Quaid-i-Azam University Faculty of Biological Sciences Fariha Hasan Quaid-i-Azam University Faculty of Biological Sciences Samiullah Khan Quaid-i-Azam University Faculty of Biological Sciences Malik Badshah Quaid-i-Azam University Faculty of Biological Sciences Sumra Wajid Abbasi National University of Medical Sciences Sajjad Ahmad Quaid-i-Azam University Faculty of Biological Sciences Ghufranud Din University of Haripur Muhammad Farman Quaid-i-Azam University Faculty of Biological Sciences Aamer Ali Shah (  alishah@qau.edu.pk ) Quaid-i-Azam University Faculty of Biological Sciences https://orcid.org/0000-0001-7454-4105 Research Article Keywords: Antioxidant, Eupatilin, Hydroxyauranetin, LC-MS, Micromonosporaaurantiaca, Radioresistant Posted Date: January 31st, 2022 Page 1/25 DOI: https://doi.org/10.21203/rs.3.rs-1277398/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/25 Abstract This study aims to determine UV-B resistance and to investigate computational analysis and antioxidant potential of methoxy- avones of Micromonospora aurantiaca TMC-15 isolated from Thal Desert, Pakistan. The cellular extract was puri ed through solid phase extraction and UV-Vis spectrum analysis indicated absorption peaks at λmax 250 nm, 343 nm, and 380 nm that revealed the presence of methoxyavones named eupatilin and 5-hydroxyauranetin. The avones were evaluated for their antioxidant as well as protein and lipid peroxidation inhibition potential using di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium (DPPH), 2,4-dinitrophenyl hydrazine (DNPH), and thiobarbituric acid reactive substances (TBARS) assays, respectively. The methoxy- avones were further studied for their docking a nity and interaction dynamics to determine their structural and energetic properties at atomic level. The antioxidant potential, protein and lipid oxidation inhibition and DNA damage preventive abilities were correlated as predicted by computational analysis. The eupatilin and 5-hydroxyauranetin binding potential to their targeted proteins 1N8Q and 1OG5 is -4.1 and -7.5 kcal/mol, respectively. Moreover, the eupatiline and 5-hydroxyauranetin complexes illustrate van der Waals contacts and strong hydrogen bonds to their respective enzymes target. Both in-vitro studies and computational analysis results revealed that methoxy- avones of Micromonospora aurantiaca TMC-15 can be used against radiation mediated oxidative damages due to its kosmotrophic nature. The demonstration of good antioxidant activities not only protect DNA but also protein and lipid oxidation and therefore could be a good candidate in radio-protective drugs and as sunscreen due to its kosmotropic nature. Introduction Low-wavelength radiation is highly mutagenic and can damage all living organisms. Some radioresistant extremophiles have adapted themselves to thrive in the presence of high levels of radiation by effective scavenging abilities of reactive oxygen and nitrogen species (Fredrickson et al., 2008). Microorganisms residing in such extreme habitats can evolve faster as compare to those living in benign environment (Li and Hong, 2016). This is associated to the high mutation rate in such harsh conditions which ultimately could lead to put selective pressure on these microbes as a result, produce something interesting of biotechnological use (Sayed et al., 2020). Diverse metabolic properties in radioresistant organisms include production of extremozymes, carotenoids, avonoids, vitamins and other potential extremolytes that can scavenge reactive radicals (Yu et al., 2015). Radioresistant extremophiles act as host for novel compounds that are not only important for their structural and biochemical diversity but also devise biotechnological applications in food, cosmetics and pharmaceutical industries. Such metabolic products not only aid in the survival of microorganisms in extreme environment but also play a role in conditions like desiccation resistance, involved in immunosuppression, enhancing antioxidant and anticancer properties, enabling oxidative damage repair (Nair et al., 2004). The exploration of radioresistant extremophiles has helped to develop bio-products for novel and improved drugs that offer protection to lipids, proteins, and nucleic acids (Waditee-Sirisattha et al., 2016). The demand of Page 3/25 radioresistant, protective biomolecules has increased in the pharmaceutical and biotechnological industries (Galano et al., 2010). Various extremolytes have been extracted from radioresistant bacteria in search for radioprotective biomolecules. Flavonoid metabolites are promising as effective antioxidant candidates (Lee et al., 2009) as they can interfere with signal transduction pathways and limit proliferation and metastasis. Flavonoids counter with reactive radicals and produce stabilized and inactive radicals (Hanasaki et al., 1994). Flavonoids can also directly scavenge free radicals. To date, there is limited information available on bioactive compounds form radiation resistant bacteria although radioresistant bacteria are ubiquitous in the environment (Beblo-Vranesevic 2017). Some of the extremophiles are also found in the genus Micromonospora, belongs to Actinobacteria. These actinobacteria have biosynthetic gene clusters, which encode various biosynthetic pathways of potentially useful metabolites (Li and Hong, 2016). Extreme environments have vast diversity of actinobacteria that could be the source of radiation protective metabolites. Actinobacteria isolated from hot spring has also been recently evaluated for bioactivities which exhibited strong antibacterial activity against many pathogenic microbes (Mehetre et al., 2019). Some studies showed that the cell wall of Micromonospora isolated form high altitude Atacama Desert soil contains meso- and hydroxy-diaminopimelic acid, and glycolipids (Carro et al., 2019). A recent study conducted on microbial colonization process on solar panel surfaces and its effect on photovoltaic e ciency also revealed the presence of extremophilic bacterial genera Deinococcus, Hymenobacter and Roseomonas and fungal Neocatenulostroma (Tanner et al., 2020). In the present study, a radiation-resistant bacterium was isolated from a soil sample of the Thal Desert. Subsequently, the growth conditions and in-vitro biological activities of avones were characterized for this radioresistant isolate. The intracellular methanol-soluble compounds from the isolated strain was extracted and puri ed for characterization and further bioactivities. The radiation-protective properties of the metabolites were screened with a view to potential biotechnological applications with emphasis on nutraceutical industry. Materials And Methods Isolation of UV-B radiation resistant strains Soil samples were collected from the Thal Desert Pakistan, diluted serially and plated on tryptone glucose yeast extract agar (TGY) containing (per liter) 5 g tryptone, 1 g glucose, 5 g yeast extract, and 1 g K2HPO4, pH 7.0. Before incubation, the plates were exposed to UV radiation for 5 min in a UV-B chamber (119× 69× 52 cm) supplied with 280 nm UV light of 20W. The rate of UV uency (energy/area/time (J/m2) for test samples was calculated by using the formula He = Ee × t where He is radiant exposure, Ee is energy of irradiance and t is the time duration maintained during radiation exposure (Sajjad et al., 2017). The survival curves were determined by sub-culturing the bacteria from the radiated plates. Bacterial survival under UV-B radiation and oxidative stress Page 4/25 The resistance of strain TMC-15 to UV-B radiation, hydrogen peroxide and mitomycin C was tested by plotting the survival curve as described previously by Mattimore and Battista (1996). The cell culture was serially diluted (1:000) optical density (OD 600) of 0.08-0.1 (McFarland standard) in sterile phosphate buffer solution (PBS) pH 7.0 and spread on TGY agar plates, followed by exposure to various doses of UV-B radiations at 280 nm and incubation for 3 to 7 days at 35°C. The survival was estimated by dividing total number of colonies on radiated plates to colonies on non-radiated (control) plates. For oxidative stress determination, strain TMC-15 (OD 600 of 0.08-0.1) was treated with 10-60 mM H2O2 and 2-10 µg/ml mitomycin-C for 30 min, followed by plating on TGY and incubation at 35°C for 3 to 7 days. The relative survival was calculated as the difference in the number of colonies between the treated and untreated cultures. All experiments for the survival curve were run in triplicate. Bacterial identi cation Molecular identi cation was carried out by extracting DNA, using a modi ed SDS based DNA extraction method (Xia et al., 2019). 16S rRNA gene sequences were then ampli ed with universal primers 27F (5’AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’). The PCR amplicons were sequenced at Macrogen Service Center (GeunChun-gu, Seoul, South Korea) and computed by using basic local alignment search tool (BLAST) and compared with homologous sequences in databases of NCBI for nearest relatives. A neighbor-joining tree was constructed using MEGA X (Kumar et al., 2018) for identi cation of strain TMC-15 and compared with already reported strains in NCBI GenBank for acquisition of accession number. The 16S rRNA sequence of strain TMC-15 was submitted to NCBI GenBank under the accession number MN721337. Extraction of intracellular bioactive compounds from strain TMC-15 Strain TMC-15 was grown under optimum conditions with continuous shaking at 150 rpm. The cells were harvested by centrifugation (5000 × g) for 15 min at 4°C, and the cell pellets were washed using PBS (pH 7.4) before placed at -4 ºC for cold shock. Methanol was added to the pellets and disrupted repeatedly by sonication until the suspension became yellow colored. The suspension was centrifuged, then passed through syringe lter (0.22 µm pore size) and nally dried out in order to remove the solvent. Characterization of bioactive compounds from strain TMC15 Fourier transform Infra-Red (FT-IR) analysis The methanolic extract was vacuum evaporated to collect the sticky material (yellow pigment) and was further subjected to drying at 30oC, the compound once puri ed using SPE C18 cartridge was subjected to FTIR analysis. The puri ed extract (10 mg sample) was mixed with 100 mg KBr for Fourier Transform Infra-Red (FTIR) analysis (Jiao et al., 2010). Absorption measurement was taken over the wavelength range of 400 to 4500 cm−1 and the spectra were recorded using a Spectrum 65 FTIR spectrometer Page 5/25 (PerkinElmer). The extract was re-dissolved in methanol and the spectrum in the range of 200-800 nm was recorded with UV-Vis spectrophotometry. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis The puri ed extract (solid phase extraction) was analyzed with an Agilent 6310 Ion Trap LC-MS. The extract was dissolved in methanol (LC-MS grade) and injected to column (C18, internal diameter 5 µm, 150 × 4.6 mm). Mobile phase was in isocratic mode consisting of acetonitrile: water (80:20) with 0.5 ml/min ow rate. The MS was used in negative ion mode to detect mass to charge (m/z) transitions [M+H]−. The data was resolved with Thermo Scienti c XcaliburTM software. Bioassays of puri ed extract from strain TMC-15 Antioxidant activity by Diphenyl-1-picrylhydrazyl (DPPH) scavenging assay The DPPH scavenging rate of the puri ed intracellular extract was measured by the method as previously described (Ebrahimzadeh et al., 2008). The reaction mixture contained 20 µl DPPH (0.1 mM) and 180 µl of the sample at different concentration (10-50 µg µl−1). The assay mixture (200 µl) was incubated in dark for 30 min at 30 ºC before the absorbance was measured at 517 nm. Ascorbic acid (10-50 µg µl−1) was used as a positive control. The relative scavenging activity was calculated using the formula [(Control absorbance - sample absorbance)/control absorbance] ×100. Iron chelation assay Iron chelation activity was determined by adding 50 µl of 2 mM ferrous chloride to 10-50 µg µl−1 of the puri ed sample (Zhao et al., 2008). The reaction was initiated by adding 20 µl of ferrozine (5 mM). The assay mixture was incubated at room temperature for 15 min in dark and the absorbance at 562 nm was measured. EDTA (10-50 µg µl−1) was a positive control and a negative control was run in parallel without any chelating agent. Protein carbonylation assay 100 µl of bovine serum albumin (BSA) was incubated with 10-50 µg µl−1 of the sample (1:1). The assay mixture received 50 µl FeSO4 (1 mM), 50 µl of H2O2 (80 mM). The mixture was incubated for 1 h at room temperature and the reaction was stopped by adding 2-3 drops of catalase. This mixture was incubated with 300 µl 2, 4-dinitrophenylhydrazine (DNPH) (10 mM) for 1 h in dark and vortexed after every 10 min. The unbound protein was precipitated by adding 50 µl of chilled 10% trichloroacetic acid (TCA). Protein precipitates were washed with 50% ethyl acetate and ethanol (1:1) (Sobeh et al., 2019). TCA was evaporated and precipitates were centrifuged at 2000 × g at 4 ºC for 10 min after adding 1 ml of guanidine hydrochloride (6 M). Absorbance of the supernatant was measured at 370 nm compared to a Page 6/25 blank run in parallel by replacing H2O2 and FeSO4 with distill water. Percentage inhibition was calculated from [(Absorbance of control - Absorbance of sample)/ Absorbance of control] ×100. Lipid peroxidation inhibition assay Thiobarbituric acid assay was performed by measuring lipid peroxidation inhibition offered by avones. 1 g rat liver tissue was fused (Bio-Gen PRO200 homogenizer) in Tris-HCl buffer at pH 7.4 and centrifuged at 3000 × g for 5 min. An aliquot (1 ml) was mixed with 10-50 µg µl−1 of the sample. The reaction was initiated by mixing 300 µl of FeSO4 (10 Mm), 200 µl sodium dodecyl sulphate (SDS) (8.1%), 250 µl acetic acid, and 250 µl thiobarbituric acid (TBA) (0.6% v/v). The assay mixture was incubated for 1 h at 100 ºC before the absorbance was measured at 532 nm. Finally, the lipid peroxidation inhibition was calculated using malondialdehyde (MDA) standard curve. DNA protection activity The DNA damage prevention assay was determined by incubating pUC18 plasmid (2 µl) treated with 10 µg µl−1 of puri ed sample, 3 µl of FeSO4 (2mM), 4 µl of H2O2 (30%), 4 µl of sodium nitroprusside (1 mM) followed by incubation for 1 h at 37ºC (Kitts et al., 2000). Double and single stranded breakage of pUC18 was generated by the attack of hydroxyl radical, observed in the negative control. In the positive control, untreated DNA was run in parallel prior to addition of any DNA damaging agents. The DNA bands of the treated extract, the negative and positive controls were separated using agarose gel electrophoresis. Computational Studies Molecular Docking Molecular docking of the compounds with representative two enzymes that respond to production of reactive oxygen species was accomplished using Autodock 4.2.6 (Morris et al., 2009). Initially, the enzymes 3D structure of the cytochrome P450 (PDB ID: 1OG5) and lipoxygenase (PDB ID: 1N8Q) were retrieved from protein data bank (Bermen et al., 2000) and subsequently treated in a short receptor preparation phase. During this phase, all co-crystalized ligands were removed from the receptors and hydrogen atoms were added. Energy minimization was performed to remove steric clashes in the enzymes structure limiting steepest descent steps to 100, conjugate gradient steps to 10 and setting the step size to 0.02 Å. AMBER ff14SB force eld was applied on the structure to treat standard residues whereas to specify net charge for the receptors AM1-BCC charge method was used. The docking procedure used in this study following protocol described by Costa et al., (2018). Before, the docking method was validated by docking the co-crystalized ligand at the same position presented in the crystal structure. For both compounds, 10 iterations were produced and ranked as per binding a nity for the receptors (binding energy in kcal/mol). Coordinates of the grid center for IN8Q on XYZ were set as: 21.864 Å, 2.184 Å and 18.909 Å whereas for 1OG5 it is -20.257 Å, 86991 Å, and 38.581 Å. The grid six points for 1N8Q were 24X, 18Y and 12Z, while for 1OG5 they were 22X,20Y and 24 Z. Conformation with high a nity (lowest binding energy) among the predicted poses was selected as complex with the enzyme to be visualized for interactions and put forward possible interaction mechanism. Page 7/25 Molecular Dynamics (MD) simulations The selection of screened small compounds for potential optimization using MD simulations depends not only on the docking energies but also on interacting residues, tness scores, pharmacophoric and low toxicity features. The study of conformational behavior of selected compounds in complex with macromolecules at atomic level is an important step in providing an insight into the structural stability, physico-chemical behavior, and biological mechanisms. In view of this, we performed a 50ns MD simulation for the prioritized complexes using AMBER 18. Using antechamber, a module embedded in Amber18, a set of initial parameters and libraries was generated (Case et al., 2008). In tleap module, ff14SB force eld for proteins and GAFF for ligands were used to investigate the stability and dynamical properties of selected compounds and dock complexes. In orthorhombic TIP3P water box at 8.0 Å, the top scoring docked conformations of all the four systems were solvated. After that, the Na+ ions were added to neutralize the systems. Energy optimization was accomplished via 2000 steps minimization of hydrogen atoms, 1000 steps minimization of system solvation box energy with the restraint of 200 kcal/mol – Å2 on the rest of the system, 1000 steps minimization of the entire set of system atoms with the restraint of 5 kcal/mol –Å2 exercised on system carbon alpha atoms, and 300 steps minimization on non-heavy atoms of the system with the restraint of 100 kcal/mol –Å2 on the remaining components of the system. The system was then heated to 300K through the NVT ensemble supported via Langevin dynamics and SHAKE algorithm to limit hydrogen bonds. An equilibration for 100-ps was attained while sustaining pressure on the system via NPT ensemble letting restraint on Cα atoms of 5 kcal/mol –Å2 (Abbasi et al., 2016; Naz et al., 2020). The resulting trajectories, using the CPPTRAJ module, were examined, for the structural stability (Roe and Cheatham 2013). MMPB/GBSA analysis for binding free energies The MMPB/GBSA method approach was further applied on the simulation trajectories to estimate free binding energy of all the four system as a difference between the receptor proteins, ligands and receptorligand complexes free energies (Miller et al., 2012). The MMPB/GBSA free binding energy is computed using equation: ΔGbind = G receptor-ligand ฀ (G receptor + G ligand) For each term, the free energy is decomposed into MM energy, polar and nonpolar solvation energy and entropy, as demonstrated by the following equation: ΔG = ΔEmolecular mechanics + ΔG solvation − T·ΔS = ΔEsum of angles + ΔEvan der Waals + ΔEelectrostatic + ΔGsolvation, polar + ΔG solvation, non-polar − T·ΔS The delta MMPB/GBSA energy of the compounds is calculated from 100 snapshots extracted from the simulation trajectories. Statistical analysis Page 8/25 Survivability of strain TMC-15 isolated from desert soil following exposure to different energy levels of ultraviolet B (UV-B) radiation. a UV radiation-resistant potential of WMA-LM9, b resistance to different concentrations of hydrogen peroxide (H2O2; mM), c resistance to different concentrations of mitomycin C (µg/ml). Percentage survivability value is calculated as N1/N0 × 100 where Ni is the value after exposure to irradiation, H2O2 or mitomycin C, and N0 is the value at time 0, for each condition tested. Results are highly signi cant among different groups at p < 0.05 (Student’s unpaired t test). Values are given as the mean ± standard deviation (SD; error bars) of triplicate experiments. Percentage scavenging activity of avones were studied by regression analysis between the ability to quench the superoxides and their respective concentrations. Single factor and two-way analysis of variance were applied for analysis of DPPH, iron chelation, lipid and protein oxidation inhibition assays. All the experiments were run in triplicates (n=3). The vertical error bars represent standard deviation values (± S.D.). Results Sample collection and screening of UVR microbes The results of bacteriological analysis of sand samples collected during summer months have shown 18 UV-resistant isolates from Thal desert. Majority of them were chromogenic where isolate TMC-15 showed high resistant to UV rays and was also able to produce pigments on the surface of TGY agar plates. Oxidative stress resistance in TMC-15 Strain TMC-15 exhibited 56% survival at 4.068×103J/m² of UV radiation (280 nm) (Fig. 1a), 53% survival in the oxidative stress test with 40 mM of H2O2 (Fig. 1b), and 52% survival at 6 µg/ml of mitomycin-C (Fig. 1). E. coli was run as control which unable to sustain on high dose of UV radiation and high concentration of H2O2, mitomycin C. survival of Strain TMC-15 gradually decreased with increase in concentration of all the three variables. Results are expressed as means ±SD and were compared using the Student’s unpaired t-test. Moreover, the percentage values had an exponential distribution. Error bars represent standard deviation for triplicate experiments. P < 0.05 is considered signi cant. Identi cation of strain TMC-15 Gram staining revealed that isolate TMC-15 is Gram positive. Biochemical characteristics revealed that strain is catalase positive, with pale orange, pointed, small colonies on TGY agar plates. 16S rRNA gene sequencing of TMC-15 demonstrated closest alignment to the phylum actinobacteria, genus Micromonospora. A phylogenetic tree constructed by Neighbor-Joining method presented 100% match of strain TMC-15 with Micromonospora aurantiaca MH588238 (Fig. 2). Characterization of cellular extract from M. aurantiaca strain TMC-15 FT-IR analysis Page 9/25 The FTIR spectra (Fig. 3) revealed peaks at 3258 cm−1 representing OH group of alcohols and peaks in the range 2921 and 2851cm−1 representing C-H groups of alkanes. The peak at 1736 cm−1 represented the presence of aromatic compounds and the 1223 and 1066 cm−1 peaks represent ether linkages (Table 2). The puri ed intracellular extract was scanned between 200 to 800 nm. The absorption spectra λmax for the puri ed fraction dissolved in methanol was noted at 343 nm, 250 nm and 380 nm. Table 1 Antioxidant activities of intracellular compound extracted in comparison to their respective standard (control) 10 20 30 40 50 70.43 72.2 74 87.3 96.1 80.2 88 91.2 95.3 97.61 34 61 66 76 87.3 65.4 72.68 76.5 89 92.9 % protein oxidation inhibition of Control (Ascorbic acid) on BSA 70.34 72.85 74.55 87.3 96.1 % protein oxidation inhibition of Control (Ascorbic acid) on mice liver 77 79.1 79.1 81.2 84 % inhibition of Control (Ascorbic acid) 80 88 91.2 95.33 97.61 % Lipid per-oxidation inhibition of Test (Intracellular extract) 25.5 33.2 42.3 55 70.2 % Lipid per-oxidation inhibition of Control (Ascorbic acid) 44 56 60.2 70 75.1 Concentration in µg ml−1 DPPH Radical Scavenging Assay % inhibition of Test (Intracellular extract) % inhibition of Control (Ascorbic acid) Iron chelation Assay % inhibition of Test (Intracellular extract) % inhibition of Control (EDTA) Protein oxidation Inhibition Assay Lipid Per-oxidation Inhibition Assay Page 10/25 Table 2 Wave number recorded, type of bond present and functional group representation in FTIR analysis. Wave Number (cm−1) Bond Functional Group 3258 O-H Alcohol 2921 C-H Alkane 2851 C-H Alkane 1736 C-H Aromatic Compound 1633 C=C Alkene 1466 C-H Alkane 1403 O-H Alcohol, Carboxylic 1223 C-O Ether 1066 C-O Ether LC-MS analysis The methanol-soluble extract exhibited a pool of peaks at a retention time of 2.8 min, with two major peaks at highest intensity (Fig. 4). The mass scan width in the negative ion mode was 100–1000. Major peaks for m/z 343.9 and 386.9 matched the formula C18H16O7 and C20H20O8, corresponding eupatilin (5,7-dihydroxy-3', 4', 6-trimethoxy avone) and hydroxyauranetin (5-hydroxy-3,6,7,8,4'pentamethoxy avone). Bioassays of puri ed extract from M. aurantiaca strain TMC-15 DPPH radical scavenging assay The avones (eupatilin and hydroxyauranetin) effect on DPPH radical scavenging is shown in Table 1, increased with concentration of the puri ed extract. 80% scavenging activity was offered by avones when used at concentration of 50 µg µl−1. Ascorbic acid was run in parallel as positive control, showed 97% scavenging activity in pure form. Lipid peroxidation and protein carbonylation assays The role of avones (eupatilin and hydroxyauranetin) against oxidative damage to lipid and protein was also studied. The oxidative effect on protein (BSA) and lipid (rat liver homogenate) was inhibited 73% and 51%, respectively. The standard ascorbic acid (50 µg µl−1) showed 79% inhibition (Table 1). Iron chelation assay Page 11/25 Oxidative damage of cellular component due to free available iron in the body is extensively linked to degenerative diseases. These free Fe+2 can generate superoxides through Fenton pathway. The electron donating potential of such compounds are also very affective in metal ion chelation as shown in Table 1 (87%) when compared with standard EDTA. DNA damage protection assay Several metabolic processes generate toxic superoxides and other active OH species. These freely available O and OH ions attack on DNA resulting oxidative damages. DNA damages have extensively studied for its relevance to pathogenesis of multiple diseases. Here, we investigated the DNA preventive effect of eupatilin and hydroxyauranetin using plasmid pUC18 with H2O2, FeSO4 and sodium nitroprusside. The gel electrophoresis results indicated that oxidative damage to plasmid pUC18 was inhibited by avones as shown in gure 5. Computational studies Molecular docking analysis The docking protocol was validated rst by docking the co-crystalized ligands at the site reported in the crystal structure. The results revealed similar compounds conformation both in docking and crystal structures thus validating the accuracy of docking protocol. For 5-hydroxyauranetin, the top docked conformation has binding a nity of -3.2 kcal mol−1 to 1N8Q and -7.0 kcal/mol for 1OG5. The eupatilin binding potential for 1N8Q and 1OG5 is -4.1 kcal mol−1 and -7.5 kcal mol−1, respectively. The interactions of compound to receptor molecules at the docked positions are provided in Figure 6. Eupatilin complexes with their target enzymes illustrates strong hydrogen bonding and intramolecular ligand Van der Waals interactions as shown in gure 7. Molecular dynamics simulation Both compounds in complex with the enzymes (1N8Q and 1OG5) were subjected to dynamics understanding. In general, all the four complexes depicted very stable behavior as demonstrated by the Cα-RMSD. This concludes strong intermolecular binding and stable conformation of the compound with the receptors. The mean Cα-RMSD computed for the complexes is: 1N8Q-5-Hydroxyyauranetin (1.7 Å), IN8Q-Eupaltin (2.2 Å), 1OG5-5-Hydroxyyauranetin (1.5 Å), and IN8Q-Eupaltin (1.8 Å). Additionally, CαRMSF of all complexes were estimated to examine residual exibility of the enzyme residues in the compound presence. In order to get insights of conformation stability, the RMSD of compounds was plotted versus simulation time. As shown in gure 8, the compounds original docked conformation is quite stable. MMPB/GBSA analysis The docking a nity of the compounds for the enzymes was tested further using popular binding energy methods of MMGBSA and MMPBSA. All the four complexes demonstrated rigorous binding energies as Page 12/25 tabulated in Table 3. To the net energy of complexes, signi cant binding energy contribution was noticed from gas phase in both MMGBSA and MMPBSA whereas the solvation energy contribution is likely to be less important in complex formation. In the solvation phase, non-polar energy somewhat plays a role in binding contrast to the polar solvation contribution. Table 3 Binding energies of the docked complexes. Method Energy Component 1N8Q-5-Hydroxyyauranetin IN8QEupaltin 1OG5-5-Hydroxyyauranetin 1OG5Eupaltin MMGBSA VDWAALS -52.82 -57.51 -42.35 -50.2933 EEL -269.83 -32.26 -30.66 -36.5203 EGB 280.48 47.40 50.64 48.5143 ESURF -7.27 -6.44 -5.57 -5.5441 DELTA G gas -322.66 -89.78 -73.02 -86.8136 DELTA G solv 273.21 40.95 45.07 42.9702 DELTA TOTAL -49.45 -48.83 -27.94 -43.8434 VDWAALS -52.82 -57.51 -42.35 -50.2933 EEL -269.83 -32.26 -30.66 -36.5203 EPB 267.61 55.64 46.66 61.2405 ENPOLAR -3.76 -3.55 -3.97 -3.7890 EDISPER 0.0000 0.0000 0.00 0.0000 DELTA G gas -322.6620 -89.7878 -73.02 -86.8136 DELTA G solv 263.8484 52.0852 42.69 57.4516 DELTA TOTAL -58.8136 -37.7026 -30.3279 -29.3621 MMPBSA Discussion Thal desert of Pakistan has been characterized by high solar radiation, salinity and wide temperature uctuation between day and night. There is no available literature to explore these extreme environments in search of new strains able to produce valuable compounds. Most of the isolates characterized in our study were able to produce colored colonies on solid agar medium. The chromogenic factor of these microbes might be helpful in their protection against UVB induced oxidative damage. Previously a number of studies have been carried out to isolate radio-resistant bacteria from desert soil, the radiation resistant property is acquired as a result of evolution that protect cells from desiccation (Rainey et al., Page 13/25 2005). The ability of these UVR microbes to survive in several extreme conditions, is suggested to be as a result of modulating gene expression in extreme conditions (Li et al., 2014) thereby producing colored compounds, shield the bacterium in stress. There is also a strong correlation between mitomycin C, H2O2 resistance and secondary metabolite production. The ability of radioresistant microbes to survive in extreme conditions is suggested to be a result of three combined mechanisms: prevention, tolerance and repair. The results also suggested that TMC-15 has a strong catalase and superoxide dismutase antioxidant system thereby protecting the cell from oxidative damages and showed prolong resistance to all three variables. The phylum actinobacteria is ubiquitous and considered one of the most valuable eco-friendly prokaryotes in eld of microbial biotechnology. Recently, methanolic extract from genus Micromonospora isolated from extreme hyper-arid Atacama Desert soil evaluated for biotechnological potential (Carro et al., 2019). There are several putative genes of Micromonospora that cope stress responses (Trujillo et al., 2014; Brettin et al., 2015) such as heat shock, cold shock, desiccation, and oxidative stress. M. aurantiaca has genes associated with SOS repair and also superoxide dismutase genes. In recent years, better understanding of ROS-induced oxidative damages and searching for new bioactive molecules and novel strategies to protect the cellular damages are the two major goals of medical research. Our results revealed that intracellular eupatilin and hydroxyauranetin, confer protection against oxidative stress conditions. There are no previous reports on eupatilin and 5-hydroxyauranetin in radio-resistant bacteria. The results from in-vitro biological activities suggested that the intracellular extract from strain TMC-15 scavenges free radicals, comparable to properties reported for radio resistant Deinococcus strains (Slade et al., 2011; Sajjad et al., 2017). The strong scavenging potential of these compounds may also be due to conjugated double bond and resonance structure which can easily transfer or donate electron to form nal stable product. The presence of keto, hydroxy groups and active fractions like quercetin and gallic acid analogs contributing to such antioxidant activities (Yang et al., 2011). Flavones have potential as chemotherapeutic drug for different pathological conditions related to free radicals. The antioxidant results offered by these avones against free radicals generated by FeSO4 in protein oxidation inhibition assay are due to the capability of such compounds to donate electrons thereby neutralizing the free radicals. In our study the avones extracted from this bacterium was more e cient in preventing the protein and lipid oxidative damages as studied in BSA and mice liver homogenate (Prazdnova et al., 2014). The kosmotrophic nature of the studied avones also aids to decrease the solubility of different cellular membranes and strengthen the intermolecular hydrogen bonding to maintain the integrity of membranes and prevent leakage of cellular contents (Krinsky and Johnson 2005). Due to conjugation double bond system, resonance structure and presence of keto groups, such compounds result in the formation of relatively stable end products which suggests that it can be effectively used in drugs and for other therapeutic applications. The biological signi cance of iron and predominantly its pathological concerns are due to its involvement in the oxidation-reduction process known as the HaberWeiss reaction where it generates different super oxides that have deleterious effects on the cells, resulting in mitochondrial damage, DNA oxidation and Page 14/25 peroxidation of membrane lipids. Moreover, the free iron can react with unsaturated lipids resulting production of alkoxyl and peroxyl radicals. The consequences of iron-overload are apparent in diseases such as thalassemia, Friedreich’s ataxia and other pathological conditions that lead to cancer (Heli et al., 2011; Kolnagou et al., 20014). Through a series of reactions these avones through oxidation-reduction mechanism form stable end products thereby prevent the cells from oxidative damage by blocking the Fenton reactions and by quenching super oxides. Iron chelation therapy signi es a novel strategy of treatment for these disorders, preventing oxidative effects by removing catalytically active iron. The gel electrophoresis results indicated that oxidative damage to plasmid pUC18 was inhibited by avones. The possible mechanism might be due to the inhibition of 7,8-oxo-2-deoxyguanosine formation generated in stress (Sajjad et al., 2017). This study also showed that many other DNA repair enzymes, cellular proteins, enzymatic antioxidants are protected by these avones. The protective ability of the studied avones contributes to prolong the cell survival in extreme conditions and signi cantly reduces the DNA damage risk (Singh and Gabani 2011). Eupatilin and 5-hydroxyauranetin have signi cant industrial and pharmaceutical importance, and now there is a potential for producing them in bioreactors. The antioxidant potential of puri ed compounds was additionally investigated computationally to decipher their binding a nity to selected receptors. Both compounds bound exactly to the same site of the enzymes and interact with common set of residues. The 5-hydroxyauranetin to 1N8Q formed two strong hydrogen bonds with Ile857 and His518 additional to strong network of hydrophobic contacts. This compound prefers to interact hydrophobically with the enzyme residues. Eupatilin complexes with their target enzymes illustrates strong hydrogen bonding and intramolecular ligand van der Waals interactions as shown in gure 7. It was observed that in the presence of compound, enzyme residues in all four complexes are signi cantly stable with exception to 1N8Q-Eupaltin where Asp20-Phe40 region showed higher uctuations. Upon inspection, uctuation in this region is the outcome of exible loop covering residue of Pro1-Asn12 and may be an approach to hold the compound strongly at the docked site. The average Cα-RMSF of the complexes is: 1N8Q-5-Hydroxyyauranetin (0.8 Å), IN8Q-Eupaltin (1.2 Å), 1OG5-5-Hydroxyyauranetin (0.8 Å), and IN8Q-Eupaltin (0.9 Å). The 1N8Q-Eupaltin as discussed earlier is showing some variation in the binding mode that might be driven by the N-terminal loop to accomplish proper intermolecular binding and enhance complex stability. For each compound, average RMSD with enzyme is: 1N8Q-5-Hydroxyyauranetin (0.5 Å), IN8Q-Eupaltin (0.7 Å), 1OG5-5-Hydroxyyauranetin (0.5 Å), and IN8Q-Eupaltin (0.4 Å).c Finally, it is concluded that Micromonospora aurantiaca strain TMC-15 isolated form extreme environment showed high rate of tolerance to UV, H2O2 and mitomycin. The avones from strain TMC-15 exhibited both antioxidant as well as oxidative damage prevention potential against cellular macromolecules. The docking analysis also revealed signi cant binding a nity of these avones to their target proteins through N-terminal loop. Both eupatilin and 5-hydroxyauranetin can be used as potential mitigator and radioprotectants in sunscreen to overcome the radiation mediated oxidative damages. Further studies are needed to determine biosynthetic pathway for enhanced production and intensi ed bioactivities through metabolic engineering. Page 15/25 Declarations Funding This work was funded by Higher Education Commission of Pakistan. Con ict of interest The authors declare no con ict of interest. Ethics approval The current study does not include any human or animal subject; therefore, the ethical committee approval is not required. Author Contributions WS, MN, TA and AAS: Preparation of the overall research plan as well as protocols for various experiments WS, MN, AR: Performed experimental work in lab as per the pre-designed research plan GD, FH, SK and MB: Facilitated in interpretation of various bioassay and phylogenetic analysis in the current research project SWA and SA: Contributed in docking and simulation analysis MF: Analysis of samples through LC-MS and interpretation of LC-MS data. WS, MN, AR, AAS: Write up of the manuscript AAS, FH, SK, MB: Proof reading of the oveall mansucript for english comprehension and typing mistakes Acknowledgements This study was supported by the Higher Education Commission of Pakistan. References 1. Abbasi S, Raza S, Azam SS, Liedl KR, Fuchs JE (2016) Interaction mechanisms of a melatonergic inhibitor in the melatonin synthesis pathway. J Mol Liq 221:507-17 2. 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Accession numbers of the sequences are shown in parentheses after the strain name. The numbers at nodes are percentage bootstrap values Figure 3 FTIR Analysis of puri ed extract isolated from strain TMC-15 Page 21/25 Figure 4 LC-MS chromatogram/negative MS spectrum of puri ed the metabolic extract. The compound was identi ed by signals at m/z of 343.9 and 386.9 [M+H]- Figure 5 DNA damage prevention assay. Lane PC represents positive control containing pUC18. Lane NC displayed negative control without any antioxidant. Sample lane contains pUC18 treated with intracellular extract from strain TMC-15. The gel photo has no letter codes (PC, NC) Page 22/25 Figure 6 Binding mode of the compounds at docked position of 1N8Q (top) and 1OG5 (bottom). The control (protocatecuic acid) in case of IN8Q (colored as plum cartoon) is shown in tan, whereas as yellow (swarfarin) in 1OG5 (colored as tan cartoon). Compounds 5-hydroxyauranetin and Eupatilin are presented by sky blue and plum, respectively in both receptors Page 23/25 Figure 7 Chemical interactions of the compounds to the proteins at the docked sites. Coloring of the interactions can be interpreted as light green (van der Waals), red (unfavorable bump), dark green (conventional hydrogen bond), aquamarine (carbon hydrogen bond), purple (pi-sigma), pink (alkyl, pi-alkyl), orange (pianion), and fuscia (pi-pi T-shaped) Page 24/25 Figure 8 MD simulation analysis. Enzymes RMSD in the presence of compounds (A), Enzymes RMSF in the presence of compounds, and compounds RMSD at the docked site of the enzymes (C) Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. Authorchecklist.docx Page 25/25