Cis‐Dihydroxylation of Tricyclic Arenes and Heteroarenes Catalyzed by Toluene Dioxygenase: A Molecular Docking Study and Experimental Validation

Toluene dioxygenase (TDO)-catalysed sulfoxidation, using Pseudomonas putida UV4, was observed for the thiophene substrates 1A-1N. The unstable thiophene oxide metabolites, 6A-6G, 6K-6N, spontaneously dimerised yielding the corresponding racemic disulfoxide cycloadducts 7A-7G, 7K-7N. Dimeric or crossed [4 + 2] cycloaddition products, derived from the thiophene oxide intermediates 6A and 6D or 6B and 6D, were found when mixtures of thiophene substrates 1A and 1D or 1B and 1D were biotransformed. The thiophene sulfoxide metabolite 6B was also trapped as cycloadducts 17 or 18 using stable dienophiles. Preferential dioxygenase-catalysed oxidation of the substituent on the thiophene ring, including exocyclic sulfoxidation (1H-1J) and cis-dihydroxylation of a phenyl substituent (1G and 1N), was also observed. An enzyme-catalysed deoxygenation of a sulfoxide in P. putida UV4 was noticed when racemic disulfoxide cyclo-adducts 7A, 7B and 7K were converted to the corresponding enantioenriched monosulfoxides 8A, 8B and 8K via a kinetic resolution process. The parent thiophene 1A and the 3-substituted thiophenes 1K-1N were also found to undergo ring dihydroxylation yielding the cis/trans-dihydrodiol metabolites 9A and 9K-9N. Evidence is provided for a dehydrogenase-catalysed desaturation of a heterocyclic dihydrodiol (9Kcis/9Ktrans) to yield the corresponding 2,3-dihydroxythiophene (24) as its preferred thiolactone tautomer (23). A simple model to allow prediction of the structure of metabolites, formed from TDO-catalysed bacterial oxidation of thiophene substrates 1, is presented.

Toluene dioxygenase (TDO)-catalysed monooxygenation of methylsulfanylmethyl phenyl sulfide 1 and methylsulfanylmethyl 2-pyridyl sulfide 4, using whole cells of Pseudomonas putida UV4, occurred exclusively at the alkyl aryl sulfur centre to yield the alkyl aryl sulfoxides 2 and 5 respectively. These sulfoxides, accompanied by the dialkyl sulfoxides 3 and 6, were also obtained from naphthalene dioxygenase (NDO)-catalysed sulfoxidation of thioacetals 1 and 4 using intact cells of P. putida NCIMB 8859. Enzymatic oxidation of methyl benzyl sulfide 7, 2-phenyl-1,3-dithiane 19, and 2-phenyl-1,3-dithiolane 23, using TDO, gave the corresponding dialkyl sulfoxides 8, 20 and 24 as minor bioproducts. TDO-catalysed dioxygenation of the alkyl benzyl sulfides 7, 15 and 17 and the thioacetals 19 and 23, with P. putida UV4, yielded the corresponding enantiopure cis-dihydrodiols 9, 16, 18, 21 and 25 as major metabolites and cis-dihydrodiol sulfoxides 14, 22 and 26 as minor metabolites, resulting from a tandem trioxygenation of substrates 7, 19 and 23 respectively. Chemical oxidation, of the enantiopure cis-dihydrodiol sulfides 9, 16, 18 and 21 with dimethyldioxirane (DMD), gave separable mixtures of the corresponding pairs of cis-dihydrodiol sulfoxide diastereoisomers 14 and 27, 28 and 29, 30 and 31, 22 and 32. While dialkyl sulfoxide bioproducts 3, 6, 20 and 24 were of variable enantiopurity (27–≥98% ee), alkyl aryl monosulfoxides 2 and 5, cis-dihydrodiols 9, 16, 18, 21 and 25 and cis-dihydrodiol sulfoxide bioproducts 14, 22 and 26 were all single enantiomers (≥98% ee). The absolute configurations of the products, obtained from enzyme-catalysed (TDO and NDO) and chemical (DMD) oxidation methods, were determined by stereochemical correlation, circular dichroism, and X-ray crystallographic methods.

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric substituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations.

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

A novel coronavirus responsible of acute respiratory infection closely related to SARS-CoV has recently emerged. So far there is no consensus for drug treatment to stop the spread of the virus. Discovery of a drug that would limit the virus expansion is one of the biggest challenges faced by the humanity in the last decades. In this perspective, to test existing drugs as inhibitors of SARS-CoV-2 main protease is a good approach. Among natural phenolic compounds found in plants, fruit, and vegetables; flavonoids are the most abundant. Flavonoids, especially in their glycosylated forms, display a number of physiological activities, which makes them interesting to investigate as antiviral molecules. The flavonoids chemical structures were downloaded from PubChem and protease structure 6LU7 was from the Protein Data Bank site. Molecular docking study was performed using AutoDock Vina. Among the tested molecules Quercetin-3-O-rhamnoside showed the highest binding affinity (-9,7 kcal/mol). Docking studies showed that glycosylated flavonoids are good inhibitors for the SARS-CoV-2 protease and could be further investigated by in vitro and in vivo experiments for further validation. MD simulations were further performed to evaluate the dynamic behavior and stability of the protein in complex with the three best hits of docking experiments. Our results indicate that the rutin is a potential drug to inhibit the function of Chymotrypsin-like protease (3CL pro) of Coronavirus.

COVID-19 (Coronavirus disease 2019) is a transmissible disease initiated and propagated through a new virus strain SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) since 31st December 2019 in Wuhan city of China and the infection has outspread globally influencing millions of people. Here, an attempt was made to recognize natural phytochemicals from medicinal plants, in order to reutilize them against COVID-19 by the virtue of molecular docking and molecular dynamics (MD) simulation study. Molecular docking study showed six probable inhibitors against SARS-CoV-2 Mpro (Main protease), two from Withania somnifera (Ashwagandha) (Withanoside V [10.32 kcal/mol] and Somniferine [9.62 kcal/mol]), one from Tinospora cordifolia (Giloy) (Tinocordiside [8.10 kcal/mol]) and three from Ocimum sanctum (Tulsi) (Vicenin [8.97 kcal/mol], Isorientin 4′-O-glucoside 2″-O-p-hydroxybenzoagte [8.55 kcal/mol] and Ursolic acid [8.52 kcal/mol]). ADMET profile prediction showed that the best docked phytochemicals from present work were safe and possesses drug-like properties. Further MD simulation study was performed to assess the constancy of docked complexes and found stable. Hence from present study it could be suggested that active phytochemicals from medicinal plants could potentially inhibit Mpro of SARS-CoV-2 and further equip the management strategy against COVID-19-a global contagion. Highlights Holistic approach of Ayurvedic medicinal plants to avenge against COVID-19 pandemic. Active phytoconstituents of Ayurvedic medicinal plants Withania somnifera (Ashwagandha), Tinospora cordifolia (Giloy) and Ocimum sanctum (Tulsi) predicted to significantly hinder main protease (Mpro or 3Clpro) of SARS-CoV-2. Through molecular docking and molecular dynamic simulation study, Withanoside V, Somniferine, Tinocordiside, Vicenin, Ursolic acid and Isorientin 4′-O-glucoside 2″-O-p-hydroxybenzoagte were anticipated to impede the activity of SARS-CoV-2 Mpro. Drug-likeness and ADMET profile prediction of best docked compounds from present study were predicted to be safe, drug-like compounds with no toxicity. Communicated by Ramaswamy H. Sarma

Bacterial strains expressing naphthalene, biphenyl, and toluene dioxygenase were examined for their abilities to oxidize 6,7-dihydro-5H-benzocycloheptene (benzocyclohept-1-ene). The major oxidation products were isolated, and their absolute configurations were determined by chiral 1H nuclear magnetic resonance analysis of diastereomeric boronate esters, chiral stationary-phase high-pressure liquid chromatography, and stereo-chemical correlation. Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae (formerly identified as a Beijerinckia sp.) B8/36 expressing naphthalene and biphenyl dioxygenases, respectively, oxidized benzocyclohept-1-ene to a major product identified as (-)-(1R,2S)-cis-dihydroxybenzocycloheptane (> 98% enantiomeric excess [ee], 50 and 90% yield, respectively). In contrast, Pseudomonas putida F39/D expressing toluene dioxygenase oxidized benzocyclohept-1-ene to (+)-(5R)-hydroxybenzocyclohept-1-ene (> 98% ee, 90% yield) as the major metabolite and to the "opposite" diol, (+)-(1S,2R)-cis-dihydroxybenzocycloheptane (> 98% ee, 10% yield). The results indicate that, for benzocyclohept-1-ene, the major reaction catalyzed by naphthalene and biphenyl dioxygenases is dioxygenation whereas toluene dioxygenase catalyzes mainly R-stereospecific benzylic monooxygenation. Although the type of reaction catalyzed by each organism was not predictable, the absolute configuration of the diol and monol products formed by naphthalene and toluene dioxygenases are consistent with the stereochemistry of the products formed by these enzymes from other benzocycloalkene substrates.

Molecular docking studies of quinoline and 2-chloroquinoline substrates at the active site of toluene dioxygenase (TDO), were conducted using Autodock Vina, to identify novel edge-to-face interactions and to rationalize the observed stereoselective cis-dihydroxylation of carbocyclic rings and formation of isolable cis-dihydrodiol metabolites. These in silico docking results of quinoline and pyridine substrates, with TDO, also provided support for the postulated cis-dihydroxylation of electron-deficient pyridyl rings, to give transient cis-dihydrodiol intermediates and the derived hydroxyquinolines. 2-Chloroquinoline cis-dihydrodiol metabolites were used as precursors in the chemoenzymatic synthesis of enantiopure arene oxide and arene dioxide derivatives of quinoline, in the context of its possible mammalian metabolism and carcinogenicity.

Synthesis of novel antipyrine-azole-S-alkyl derivatives antimicrobial activity, molecular docking, and computational studies

Antifungal resistance represents a major challenge for treating invasive fungal infections due to the limited arsenal of systemic antifungal agents. Azole resistance among Candida and Aspergillus species and the spread of such species is alarming. Isoxazoles play a significant role in the field of medicinal chemistry. The great interest associated with this class of compounds is their versatility as synthetic intermediates. Isoxazole derivatives were found to possess anti-bacterial, anti-fungal, anti-inflammatory, pesticidal and herbicidal activities. This study focuses on screening some novel isoxazoles against essential enzymes of C. albicans using molecular docking and dynamic studies. Molecular docking studies were performed on twenty novel isoxazoles against N- methyl transferase and DNA gyrase of C. albicans using GLIDE. 2D and 3D dock poses were visualized. Molecular dynamic study was carried out by DESMOND. Docking study in the XP mode has given significant results with good GScores and Emodel for NMT. Compound 2D (2-hydroxy derivative) was ranked highest among the derivatives. The ligands bound tightly with the target protein, indicated by of hydrogen bond interactions and pi-pi stacking interactions at the active site. Molecular dynamic studies were carried out for two complexes. Complex was found to be stable during molecular dynamic simulation. Docking study suggests that compound 2D (2-hydroxy derivative) may be prospective inhibitors of N-methyl transferase as it are specific in binding to the active site of this enzyme. Hence it may be considered as lead molecule for the design of potential inhibitors of C. albicans.

BackgroundArdisia gigantifolia Stapf. (AGS), a Chinese folk medicine widely grows in the south of China and several studies reported that AGS could inhibit the proliferation of breast cancer, liver cancer, and bladder cancer cell lines. However, little is known about its anti-colorectal cancer (CRC) efficiency.MethodsIn the present study, a combination of MTT assay, network pharmacological analysis, bioinformatics, molecular docking, and molecular dynamics simulation study was used to investigate the active ingredients, and targets of AGS against CRC, as well as the potential mechanism.ResultsMTT assay showed that three kinds of fractions from AGS, including the n-butanol extract (NBAGS), ethyl acetate fraction (EAAGS), and petroleum ether fraction (PEAGS) significantly inhibited the proliferation of CRC cells, with the IC50 values of 197.24, 264.85, 15.45 µg/mL on HCT116 cells, and 523.6, 323.59, 150.31 µg/mL on SW620 cells, respectively. Eleven active ingredients, including, 11-O-galloylbergenin, 11-O-protocatechuoylbergenin, 11-O-syringylbergenin, ardisiacrispin B, bergenin, epicatechin-3-gallate, gallic acid, quercetin, stigmasterol, stigmasterol-3-o-β-D-glucopyranoside were identified. A total of 173 targets related to the bioactive components and 21,572 targets related to CRC were picked out through database searching. Based on the crossover targets of AGS and CRC, a protein-protein interaction network was built up by the String database, from which it was concluded that the core targets would be SRC, MAPK1, ESR1, HSP90AA1, MAPK8. Besides, GO analysis showed that the numbers of biological process, cellular component, and molecular function of AGS against CRC were 1079, 44, and 132, respectively, and KEGG pathway enrichment indicated that 96 signaling pathways in all would probably be involved in AGS against CRC, among which MAPK signaling pathway, lipid, and atherosclerosis, proteoglycans in cancer, prostate cancer, adherens junction would probably be the major pathways. The docking study verified that AGS had multiple ingredients and multiple targets against CRC. Molecular dynamics (MD) simulation analysis showed that the binding would be stable via forming hydrogen bonds.ConclusionOur study showed that AGS had good anti-CRC potency with the characteristics of multi-ingredients, -targets, and -signaling pathways.

A comparative study of volatiles, antioxidant activity, phytotoxic activity, as well as in silico molecular docking and ADMET study, was conducted for essential oils from three Vitex species, viz., V. agnus-castus, V. negundo, and V. trifolia. Essential oils (OEs) extracted by hydrodistillation were subjected to compositional analysis using GC-MS. A total number of 37, 45, and 43 components were identified in V. agnus-castus, V. negundo, and V. trifolia, respectively. The antioxidant activity of EOs, assessed using different radical-scavenging (DPPH, H2O2 and NO), reducing power, and metal chelating assays, were found to be significant as compared with those of the standards. The phytotoxic potential of the EOs was performed in the receptor species Raphanusraphanistrum (wild radish) and the EOs showed different levels of intensity of seed germination inhibition and root and shoot length inhibition. The molecular docking study was conducted to screen the antioxidant and phytotoxic activity of the major and potent compounds against human protein target, peroxiredoxin 5, and 4-hydroxyphenylpyruvate dioxygenase protein (HPPD). Results showed good binding affinities and attributed the strongest inhibitory activity to 13-epi-manoyl oxide for both the target proteins.

Background In the present study, we investigated the antibacterial, anthelmintic, and analgesic activities of methanol extract of P. sylvaticum leaves (MEPSL) in experimental models. Then, computational analysis (in silico molecular docking and PASS prediction) was performed to determine the potent phytoconstituents of total six isolated compounds of this plant for antibacterial and anthelmintic activities. Methods Qualitative and quantitative phytochemical studies were carried out by established methods. In vitro antibacterial activity was determined by disc diffusion technique and anthelmintic activity was tested against Tubifex tubifex worm whereas analgesic activity was determined by the acetic acid-induced writhing test in mice. Molecular docking study was performed using Schrödinger Maestro 10.1 and an online tool used for PASS prediction. Results Our phytochemical study revealed the presence of alkaloids, flavonoids, saponins, and also indicated a substantial amount of phenols (65.83 mg), flavonoids (102.56 mg), and condensed tannins (89.32 mg). MEPSL showed good antibacterial activity against both gram-positive and gram-negative bacteria. Our result exhibited that MEPSL has strong anthelmintic action compared to standard levamisole. In addition, the extract also showed a dose-dependent and statistically significant analgesic activity at the doses of 200 and 400 mg/kg, body weight. Docking studies showed that piperine and piperlonguminine have the best scores for the tested enzymes. PASS predicted the antibacterial and anthelmintic activity of both phytoconstituents. Conclusions This study suggests that MEPSL possess significant antibacterial, anthelmintic, and analgesic activities which could be related to the presence of several phytochemicals. The phytoconstituents, i.e. piperine and piperlonguminine were found to be most effective in computational studies.

Kinesin Eg5 plays an essential role in the early stages of mitosis, and it is an interesting drug target for the design of potent inhibitors. In this work, combined molecular modeling studies of molecular docking, receptor-guided QSAR methodology, and molecular dynamics (MD) simulation were performed on a series of novel S-trityl-l-cysteine (STLC) analogues as Eg5 inhibitors to understand the structural features and key residues which are involved in the inhibition. Molecular docking study was used to find the actual conformations of STLC analogues in the binding site of Eg5. Multiple linear regression (MLR), artificial neural network (ANN), and support vector machine (SVM) models were developed by the conformation which was obtained by performing docking studies. The satisfactory result of the SVM model (R2 = 0.962, SE = 0.210, RMSE = 0.190, and Q2LOO = 0.930) demonstrated the superiority of this model over other models. Also, the satisfactory agreement between experiment and predicted inhibitory values suggested that the SVM model represents good correlation and predictive power. Molecular docking was used to study the functionalities of active molecular interaction between inhibitors and Eg5. Moreover, molecular dynamics (MD) simulation was performed on the best inhibitor-Eg5 complex to investigate the stability of docked conformation and to study the binding interactions in detail. The MD simulation result showed four hydrogen bond interactions with Eg5 residues including Gly117, Glu116, Gly117, and Glu118. The outcome of this study can be used as a guideline to better interpret the protein-ligand interaction and also can assist in the designing and development of more potent Eg5 inhibitors.

The present study aimed to identify new selective inhibitors for acetylcholinesterase, butyrylcholinesterase, monoacylglycerol lipase, beta-secretase, and Asparagine endopeptidase, the targets enzymes in Alzheimer's disease. The inhibitory effect of P. atlantica Desf. methanol extracts against AChE were determined using Ellman's method. The molecular docking study is achieved using Autodock Vina. The structures of the molecules 3-methoxycarpachromene, masticadienonic acid, 7-ethoxycoumarin, 3',5,7- trihydroxy-4'-methoxyflavanone and 5,6,7,4'-tetrahydroxyflavonol-3-O-rutinoside and the five enzymes were obtained from the PubChem database and Protein databank. ADMET parameters were checked to confirm their pharmacokinetics using swiss-ADME and ADMET-SAR servers. P. atlantica Desf. methanol extracts showed a notable inhibitory effect against AChE (IC50 = 0.26 ± 0.004 mg/ml). The molecular docking results of 3-methoxycarpachromene, masticadienonic acid, 7-ethoxycoumarin, 3',5,7-trihydroxy-4'-methoxyflavanone and 5,6,7,4'-tetrahydroxyflavonol-3-Orutinoside with the five enzymes show significant affinities of these molecules towards Alzheimer disease targets, where they could form several interactions, such as hydrogen bonds and hydrophobic interactions with the studied enzymes. The shortest hydrogen bond is 1.7 A° between masticadienonic acid and Arg128 of the active site of BACE, while the lowest free energy is -11.2 of the complex 5,6,7,4'-tetrahydroxyflavonol-3-O-rutinoside -HuBchE. To the best of our knowledge, these molecules' potential anti-Alzheimer disease effect is studied in this paper for the first time. The docking studies of this work show that 3-methoxycarpachromene and masticadienonic acid, 7-ethoxycoumarin, 3',5,7-Trihydroxy-4'-methoxyflavanone and 5,6,7,4'-tetrahydroxyflavonol- 3-O-rutinoside have good affinities towards the enzymes involved in Alzheimer pathology, which confirm the ability of these molecules to inhibit the studied enzymes namely: HuAChE, HuBChE, BACE, MAGL, and AEP. These molecules might become drug candidates to prevent Alzheimer's disease.