An Antifungal Chitosanase from Bacillus subtilis SH21

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC–MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.     

Synthesis,characterization, and in silico molecular docking study of bidentate hydroxy α-aminophosphonates derivative and its Mn(II), Fe(III) and Zn(II) metal complexes as potent antioxidant and antibacterial agents

A bidentate hydroxy α-aminophosphonates ligand, diethyl-(2-hydroxyphenyl) (phenylamino)methylphosphonate and its Mn(II), Fe(III), and Zn(II) metal complexes were prepared in the molar ratio of 2:1 and characterized by spectral, thermal, analytical and physicochemical methods including UV–Vis, FT-IR, molar conductance, AAS, elemental analysis, ¹H NMR and PXRD. Thermogravimetric analysis (TG/DTG) was done to investigate the thermal decomposition/mass loss behavior of the metal complexes. Antibacterial activity was checked against 2 g-positive (B. subtilis and S. aureus) and 2 g-negative (S. typhi and E. coli) bacteria with different concentrations of compounds (250–1000 μg/ml), where Amikacin was used as a standard. The antioxidant capacity of prepared compounds was checked against DPPH and FRAP assay, where BHT was used as standard. In-silico molecular docking was also studied by Autodock 4.2 software for all compounds with S. typhi cell membrane protein OmpF complex (PDB ID- 4KR4) in which good interaction was observed for all the docked compounds.     

Identification of Antibacterial Metabolites from Endophytic Fungus Aspergillus fumigatus,Isolated from Albizia lucidior Leaves (Fabaceae), Utilizing Metabolomic and Molecular Docking Techniques

The rapid spread of bacterial infection caused by Staphylococcus aureus has become a problem to public health despite the presence of past trials devoted to controlling the infection. Thus, the current study aimed to explore the chemical composition of the extract of endophytic fungus Aspergillus fumigatus, isolated from Albizia lucidior leaves, and investigate the antimicrobial activity of isolated metabolites and their probable mode of actions. The chemical investigation of the fungal extract via UPLC/MS/MS led to the identification of at least forty-two metabolites, as well as the isolation and complete characterization of eight reported metabolites. The antibacterial activities of isolated metabolites were assessed against S. aureus using agar disc diffusion and microplate dilution methods. Compounds ergosterol, helvolic acid and monomethyl sulochrin-4-sulphate showed minimal inhibitory concentration (MIC) values of 15.63, 1.95 and 3.90 µg/mL, respectively, compared to ciprofloxacin. We also report the inhibitory activity of the fungal extract on DNA gyrase and topoisomerase IV, which led us to perform molecular docking using the three most active compounds isolated from the extract against both enzymes. These active compounds had the required structural features for S. aureus DNA gyrase and topoisomerase IV inhibition, evidenced via molecular docking.     

Identification of Potential Inhibitors of MurD Enzyme of Staphylococcus aureus from a Marine Natural Product Library

Staphylococcus aureus is an opportunistic pathogen that can cause fatal bacterial infections. MurD catalyzes the formation of peptide bond between UDP-N-acetylehyl-l-alanine and d-glutamic acid, which plays an important role in the synthesis of peptidoglycan and the formation of cell wall by S. aureus. Because S. aureus is resistant to most existing antibiotics, it is necessary to develop new inhibitors. In this study, Schrodinger 11.5 Prime homology modeling was selected to prepare the protein model of MurD enzyme, and its structure was optimized. We used a virtual screening program and similarity screening to screen 47163 compounds from three marine natural product libraries to explore new inhibitors of S. aureus. ADME provides analysis of the physicochemical properties of the best performing compounds during the screening process. To determine the stability of the docking effect, a 100 ns molecular dynamics was performed to verify how tightly the compound was bound to the protein. By docking analysis and molecular dynamics analysis, both 46604 and 46608 have strong interaction with the docking pocket, have good pharmacological properties, and maintain stable conformation with the target protein, so they have a chance to become drugs for S. aureus. Through virtual screening, similarity screening, ADME study and molecular dynamics simulation, 46604 and 46608 were selected as potential drug candidates for S. aureus.     

Purification and Partial Characterization of Bacillomycin L Produced by Bacillus amyloliquefaciens K103 from Lemon

Bacillus amyloliquefaciens K103 isolated from a lemon sample was used as a biocontrol agent to suppress Rhizoctonia solani Kühn and other fungal plant pathogens. Two antifungal compounds were purified from the culture broth using acid precipitation, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis indicated that the antifungal compounds were two isomers similar to bacillomycin L. One of the predominant active fractions was subjected to quadrupole time-of-flight mass spectrometry and amino acid analysis to determine its structural characteristics, revealing that the antifungal compound with a molecular mass of 1,034.5464 was identical to bacillomycin L. This is the second report of lemon microflora producing bacillomycin L or any antifungal compound, suppressing the growth of R. solani Kühn. Meanwhile, the study provided insights into the enormous potential of food microbial resources and bacillomycin L antibiotics in biological control and sustainable agriculture.     

Fusopoltides B–E,new polyketides isolated from Fusarium solani B-18

Four new polyketides, fusopoltides B–E, were isolated, along with four known compounds, from Fusarium solani B-18 cultured on brown-rice. Fusopoltide B is a diastereomer of its co-isolated known compound, fusopoltide A, featuring a pentaleno[1,2-c]pyran ring system. Fusopoltide C and fusopoltides D–E are incorporated the first natural polyketides featured decahydro-pentaleno[1,2-c]pyran and pentaleno[1,2-c]furan ring systems, respectively. The structural assignments were established using comprehensive spectroscopic techniques, the modified Mosher's ester method, and electronic circular dichroism (ECD) calculations.     

Prediction of COVID-19 manipulation by selective ACE inhibitory compounds of Potentilla reptant root: In silico study and ADMET profile

In the novel SARS-CoV-2 (COVID-19) as a global emergency event, the main reason of the cardiac injury from COVID-19 is angiotensin-converting enzyme 2 (ACE2) targeting in SARS-CoV-2 infection. The inhibition of ACE2 induces an increase in the angiotensin II (Ang II) and the angiotensin II receptor type 1 (AT1R) leading to impaired cardiac function or cardiac inflammatory responses. The ethyl acetate fraction of Potentilla reptans L. root can rescue heart dysfunction, oxidative stress, cardiac arrhythmias and apoptosis. Therefore, isolated components of P. reptans evaluated to identify natural anti-SARS-CoV-2 agents via molecular docking.In silico molecular docking study were carried out using the Auto Dock software on the isolated compounds of Potentilla reptans root. The protein targets of selective ACE and others obtained from Protein Data Bank (PDB). The best binding pose between amino acid residues involved in active site of the targets and compounds was discovered via molecular docking. Furthermore, ADMET properties of the compounds were evaluated.The triterpenoids of P. reptans showed more ACE inhibitory potential than catechin in both domains. They were selective on the nACE domain, especially compound 5. Also, the compound 5 & 6 had the highest binding affinity toward active site of nACE, cACE, AT1R, ACE2, and TNF-α receptors. Meanwhile, compound 3 showed more activity to inhibit TXA2. Drug likeness and ADMET analysis showed that the compounds passed the criteria of drug likeness and Lipinski rules. The current study depicted that P. reptans root showed cardioprotective effect in COVID-19 infection and manipulation of angiotensin II-induced side effects.     

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.     

Novel Fluorinated 7-Hydroxycoumarin Derivatives Containing an Oxime Ether Moiety: Design,Synthesis, Crystal Structure and Biological Evaluation

A series of fluorinated 7-hydroxycoumarin derivatives containing an oxime ether moiety have been designed, synthesized and evaluated for their antifungal activity. All the target compounds were determined by ¹H-NMR, ¹³C-NMR, FTIR and HR-MS spectra. The single-crystal structures of compounds 4e, 4h, 5h and 6c were further confirmed using X-ray diffraction. The antifungal activities against Botrytis cinerea (B. cinerea), Alternaria solani (A. solani), Gibberella zeae (G. zeae), Rhizoctorzia solani (R. solani), Colletotrichum orbiculare (C. orbiculare) and Alternaria alternata (A. alternata) were evaluated in vitro. The preliminary bioassays showed that some of the designed compounds displayed the promising antifungal activities against the above tested fungi. Strikingly, the target compounds 5f and 6h exhibited outstanding antifungal activity against B. cinerea at 100 μg/mL, with the corresponding inhibition rates reached 90.1 and 85.0%, which were better than the positive control Osthole (83.6%) and Azoxystrobin (46.5%). The compound 5f was identified as the promising fungicide candidate against B. cinerea with the EC₅₀ values of 5.75 μg/mL, which was obviously better than Osthole (33.20 μg/mL) and Azoxystrobin (64.95 μg/mL). Meanwhile, the compound 5f showed remarkable antifungal activities against R. solani with the EC₅₀ values of 28.96 μg/mL, which was better than Osthole (67.18 μg/mL) and equivalent to Azoxystrobin (21.34 μg/mL). The results provide a significant foundation for the search of novel fluorinated 7-hydroxycoumarin derivatives with good antifungal activity.     

Pharmacophore modeling,virtual screening and molecular docking of ATPase inhibitors of HSP70

Heat shock protein 70 is an effective anticancer target as it influences many signaling pathways. Hence the study investigated the important pharmacophore feature required for ATPase inhibitors of HSP70 by generating a ligand based pharmacophore model followed by virtual based screening and subsequent validation by molecular docking in Discovery studio V4.0. The most extrapolative pharmacophore model (hypotheses 8) consisted of four hydrogen bond acceptors. Further validation by external test set prediction identified 200 hits from Mini Maybridge, Drug Diverse, SCPDB compounds and Phytochemicals. Consequently, the screened compounds were refined by rule of five, ADMET and molecular docking to retain the best competitive hits. Finally Phytochemical compounds Muricatetrocin B, Diacetylphiladelphicalactone C, Eleutheroside B and 5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]amino}phenyl)- 4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic acid were obtained as leads to inhibit the ATPase activity of HSP70 in our findings and thus can be proposed for further in vitro and in vivo evaluation.     

Synthesis,characterization, docking and antimicrobial studies of binol based amide linked symmetrical bistriazoles

The antimicrobial resistance and excessive use of modern drugs has hampered the treatment of infectious disease. Therefore, the development of new antibiotics with very good efficacy and lesser side effects is the focused area of medicinal chemists. In the present work, the design and copper (I) catalyzed click synthesis of some amide linked binol based 1,4-disubstituted 1,2,3-bistriazole hybrids from bisalkyne precursor is reported along with characterization of the newly synthesized bistriazoles with the support of IR, NMR and HRMS spectral techniques. The in vitro antimicrobial screening of these bistriazoles against selected bacterial (S. aureus, B. subtilis, and E. coli) and fungal (A. niger and C. albicans) strains signifies the importance of amide and triazole moiety in the final derivatives. The in silico docking studies further supports the antimicrobial results through various binding interactions with binding energies of compound 6f (1KZN: 9.7 kcal/mol) and 6h (4WMZ: 13,3 kcal/mol).     

Molecular Docking and 3D QSAR Research of Indolocarbazole Series as Cyclin-Dependent Kinase Inhibitors

Fifty indolocarbazole series as cyclin-dependent kinase inhibitors (CDKs) are used to establish a threedimensional quantitative structure-activity relationship (3D QSAR) model based on docking conformations resulting from the Topomer comparative molecular field analysis (Topomer CoMFA). The statistic parameters show that the cross-validation (q²), the multiple correlation coefficient of fitting (r²), and external validation statistic (Q_ext²) are 0.953, 0.968, and 0.954, respectively. It is demonstrated that this Topomer CoMFA model has good stability and prediction ability. The methodology of the fragment-based drug design (FBDD) was also used to virtually screen new CDKs by the Topomer Search technology. Four similar substitutional groups selected from the ZINC database were added to the basic scaffold. As a result, 18 new CDKs with high activities were obtained. The template molecule and new designed compounds are used to study the binding relationship between the ligands and the receptor protein with Surflex-Dock. The docking results suggest good binding interactions of the designed compounds with protein. There are several hydrogen bondings between CDKs with amino acid residues of LYS33, LYS89, ASP86, LEU83, GLU81.     

Valorization of Okara by Enzymatic Production of Anti-Fungal Compounds for Plant Protection

Okara is a soybean transformation agri-food by-product, the massive production of which currently poses severe disposal issues. However, its composition is rich in seed storage proteins, which, once extracted, can represent an interesting source of bioactive peptides. Antimicrobial and antifungal proteins and peptides have been described in plant seeds; thus, okara is a valuable source of compounds, exploitable for integrated pest management. The aim of this work is to describe a rapid and economic procedure to isolate proteins from okara, and to produce an enzymatic proteolyzed product, active against fungal plant pathogens. The procedure allowed the isolation and recovery of about 30% of okara total proteins. Several proteolytic enzymes were screened to identify the proper procedure to produce antifungal compounds. Antifungal activity of the protein digested for 24 h with pancreatin against Fusarium and R. solani mycelial growth and Pseudomonas spp was assessed. A dose-response inhibitory activity was established against fungi belonging to the Fusarium genus. The exploitation of okara to produce antifungal bioactive peptides has the potential to turn this by-product into a paradigmatic example of circular economy, since a field-derived food waste is transformed into a source of valuable compounds to be used in field crops protection.     

Synthesis,thermal property and antifungal evaluation of pyrazine esters

Synthesis, thermal properties, as well as antifungal assessment of pyrazine esters were handed in this work. The metal-free esterification of pyrazinyl methanol and acid chloride derivatives in a stoichiometric ratio yielded the following compounds: pyrazin-2-ylmethyl benzoate (3a), (5-methylpyrazin-2-yl) methyl benzoate (3b), 1-(pyrazin-2-yl) ethyl benzoate (3c), 2-(pyrazin-2-yl) ethyl benzoate (3d), pyrazin-2-ylmethyl pivalate (3e). The spectral results totally showed that the synthesis conditions used permitted the compounds to be produced with great yield. The characterization included ¹H NMR, ¹³C NMR, IR, HRMS and organoleptic tests. Their thermal degradation process was examined by using the TG (thermogravimetry) and DSC (differential scanning calorimeter) methods. The pyrolysis products of the pyrazine esters in inert and oxidative atmospheres were analyzed by the Py-GC/MS (pyrolysis–gas chromatography/mass spectrometry) method. It revealed that 3a–3e were totally evaporated throughout the pyrolysis experiments at low temperatures. And they evaporated with high relative contents in pure nitrogen (86.12%–94.17%) and oxidative (75.01%–88.85%) atmospheres, with only a small amount decomposing into a series of substances. Additionally, the in vitro antifungal effects of compounds 3a–3e against R. solani, P. nicotianae, F. oxysporum, F. graminearum, and F. moniliforme were further investigated using the mycelial growth rate methodology. The findings shown that compound 3c has 94% and 80% inhibitor rates at 0.5 mg/ml against R. solani and P. nicotianae, respectively, with an EC₅₀ value (half maximum effective concentration) of 0.0191 mg/ml and 0.1870 mg/ml, respectively. Similarly, that of compounds 3a and 3b exhibited 82% and 91% at 0.5 mg/ml against R. solani, with an EC₅₀ value of 0.0209 mg/ml and 0.0218 mg/ml, respectively. The molecular docking of compound 3c with SDH (Succinate dehydrogenase) was preliminarily performed to reveal the binding modes in active pocket and analyze the interactions between the molecules and the protein.     

Synthesis and Antibacterial Study of Novel Harmine Derivatives and Tetrahydro-β-Carboline Derivatives In Vitro

Dairy mastitis is a disease of dairy cattle caused by a variety of pathogenic microorganisms which has biought huge economic losses aused huge economic losses to the world. In this paper, Harmine derivatives and tetrahydro-β-carboline derivatives synthesized by the splice method are shown to have a good inhibitory effect on the pathogenic bacteria of dairy mastitis. The results of a bacteriostatic test on pathogenic bacteria of dairy cow mastitis (S. dysgalactiae, S. pyogenes, B. subtilis and P. vulgaris) showed that compound 7l had the best bacteriostatic effect on Streptococcus dysgalactiae, with a mic value of 43.7 μ g/mL. When the concentration of 7l was 1 × MIC and 2 × MIC, it had a significant inhibitory effect on Streptococcus dysgalactiae, and there was almost no growth of Streptococcus dysgalactiae at 4 × MIC. The binding properties of target compound 7l to amine oxidase [flavin-containing] A protein were simulated by the molecular docking technique. The ligand 7l achieved strong binding with the receptor through three hydrogen bonds. The hydrogen bonds were amino acid residues thr-52, arg-51 and ser-24, which are the main force for the compound to bind to active sites.     

Molecular modeling and docking studies of new antimicrobial antipyrine-thiazole hybrids

A series of new antipyrine incorporated thiazole derivatives having phenoxyacetamide moiety as a link bridge was synthesized. The synthetic strategy involves condensation of the precursor N-(4-antipyrinyl)-2-(4-formylphenoxy)acetamide with thiosemicarbazide followed by heterocyclization of the produced thiosemicarbazone with various α-halogenated carbonyl compounds (namely; 4-chlorophenacyl bromide, ethyl bromoacetate, 3-chloroacetylacetone and ethyl 4-chloroacetoacetate). Moreover, the quantum chemical calculations at DFT/B3LYP level were used to determine the HOMO-LUMO energies and Fukui’s indices toward nucleophilic, electrophilic and radical attacks. The investigated compounds were arranged due to HOMO-LUMO energy gap as following 6 < 5 < 7 < 3 < 2 < 4 < 8. The synthesized antipyrinyl-thiazole hybrids were screened to evaluate their antibacterial and antifungal efficacies. Using Chloramphenicol as reference material, the synthesized antipyrinyl-thiazole hybrids were revealed a remarkable activity against S. aureus than B. subtilis, as example for Gram’s positive strains. The antipyrine-thiazole compounds 3, 4, 6 and 8 exhibited significant MIC values. However, the antipyrine-thiazole hybride 4 displayed reputable activities against Gram’s negative strains S. typhimurium and E. coli, respectively, in comparison with Cephalothin. Likewise, the compounds 7 and 8 were demonstrated respectable antifungal efficacy toward C. albicans in contrast to cycloheximide grade. The theoretical molecular docking studies were applied to simulate reactivity of the synthesized antipyrine-thiazole hybrids against contrasting binding sites for both of Staphylococcus aureus “Homo sapiens” (pdb: 3HUN) protein and E.coli “Homo sapiens” (PDB: 2EXB) protein. The theoretical and practical antibacterial and antifungal activities result in this work designated a proper agreement.     

Targeting 3CLpro and SARS-CoV-2 RdRp by Amphimedon sp. Metabolites: A Computational Study

Since December 2019, novel coronavirus disease 2019 (COVID-19) pandemic has caused tremendous economic loss and serious health problems worldwide. In this study, we investigated 14 natural compounds isolated from Amphimedon sp. via a molecular docking study, to examine their ability to act as anti-COVID-19 agents. Moreover, the pharmacokinetic properties of the most promising compounds were studied. The docking study showed that virtually screened compounds were effective against the new coronavirus via dual inhibition of SARS-CoV-2 RdRp and the 3CL main protease. In particular, nakinadine B (1), 20-hepacosenoic acid (11) and amphimedoside C (12) were the most promising compounds, as they demonstrated good interactions with the pockets of both enzymes. Based on the analysis of the molecular docking results, compounds 1 and 12 were selected for molecular dynamics simulation studies. Our results showed Amphimedon sp. to be a rich source for anti-COVID-19 metabolites.     

Fusaspirols A-D,novel oxaspirol derivatives isolated from Fusarium solani B-18

Four novel compounds with γ-methylidene-spirobutanolide core, fusaspirols A-D, were isolated from the brown rice culture of Fusarium solani B-18. Their structures were established by extensive spectroscopic analyses of 1D/2D-NMR, HRESITOFMS, and chemical derivatization. The absolute configurations of secondary alcohols in fusaspirols A and D were determined using modified Mosher's ester method. Fusaspirol A and 4,9-di-O-acetylfusaspirol A activated a signaling pathway in osteoclastic differentiation of murine macrophage derived RAW264.7?cells.