Identification of SARS-CoV-2 RNA-dependent RNA polymerase inhibitors from the major phytochemicals of Nigella sativa: An in silico approach

The coronavirus disease 2019 (COVID-19), which emerged in December 2019, continues to be a serious health concern worldwide. There is an urgent need to develop effective drugs and vaccines to control the spread of this disease. In the current study, the main phytochemical compounds of Nigella sativa were screened for their binding affinity for the active site of the RNA-dependent RNA polymerase (RdRp) enzyme of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The binding affinity was investigated using molecular docking methods, and the interaction of phytochemicals with the RdRp active site was analyzed and visualized using suitable software. Out of the nine phytochemicals of N. sativa screened in this study, a significant docking score was observed for four compounds, namely α-hederin, dithymoquinone, nigellicine, and nigellidine. Based on the findings of our study, we report that α-hederin, which was found to possess the lowest binding energy (–8.6 kcal/mol) and hence the best binding affinity, is the best inhibitor of RdRp of SARS-CoV-2, among all the compounds screened here. Our results prove that the top four potential phytochemical molecules of N. sativa, especially α-hederin, could be considered for ongoing drug development strategies against SARS-CoV-2. However, further in vitro and in vivo testing are required to confirm the findings of this study.     

Screening of inhibitors against SARS-CoV-2 spike protein and their capability to block the viral entry mechanism: A viroinformatics study

SARS-CoV-2, previously named 2019 novel coronavirus (2019-nCoV), has been associated with the global pandemic of acute respiratory distress syndrome. First reported in December 2019 in the Wuhan province of China, this new RNA virus has several folds higher transmission among humans than its other family member (SARS-CoV and MERS-CoV). The SARS-CoV-2 spike receptor-binding domain (RBD) is the region mediating the binding of the virus to host cells via Angiotensin-converting enzyme 2 (ACE2), a critical step of viral. Here in this study, we have utilized in silico approach for the virtual screening of antiviral library extracted from the Asinex database against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein. Further, the molecules were ranked based on their binding affinity against RBD, and the top 15 molecules were selected. The affinity of these selected molecules to interrupt the ACE2-Spike interaction was also studied. It was found that the chosen molecules were demonstrating excellent binding affinity against spike protein, and these molecules were also very effectively interrupting the ACE2-RBD interaction.Furthermore, molecular dynamics (MD) simulation studies were utilized to investigate the top 3 selected molecules' stability in the ACE2-RBD complexes. To the best of our knowledge, this is the first study where molecules' inhibitory potential against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein and their inhibitory potential against the ACE2-Spike has been studied. We believe that these compounds can be further tested as a potential therapeutic option against COVID-19.     

Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).     

A metabolomics approach to investigate the proceedings of mitochondrial dysfunction in rats from prediabetes to diabetes

BackgroundDiabetes mellitus (DM) is a leading cause of preventable cardiovascular disease, but the metabolic changes from prediabetes to diabetes have not been fully clarified. This study implemented a metabolomics profiling platform to investigate the variations of metabolites and to elucidate their global profiling from metabolic syndrome to DM. Methods: Male Sprague-Dawley rats (n = 44) were divided into four groups. Three groups were separately fed with a normal diet, a high-fructose diet (HF), or a high-fat (HL) diet while one group was treated with streptozotocin. The HF and HL diet were meant to induce insulin resistance, obesity, and dyslipidemia, which known to induce DM. Results: The most significant metabolic variations in the DM group’s urine samples were the reduced release of citric acid cycle intermediates, the increase in acylcarnitines, and the decrease in urea excretion, all of which indicated energy metabolism abnormalities and mitochondrial dysfunction. Overall, the metabolic analysis revealed tryptophan metabolic pathway variations in the prediabetic phase, even though the mitochondrial function remains unaffected. Conclusion: This study show that widespread methylations and impaired tryptophan metabolism occur in metabolic syndrome and are then followed by a decline in citric acid cycle intermediates, indicating mitochondrial dysfunction in diabetes.     

N-phenyl ureidobenzenesulfonates,a novel class of promising human dihydroorotate dehydrogenase inhibitors

N-phenyl ureidobenzenesulfonates (PUB-SOs) is a new class of promising anticancer agents inducing replication stresses and cell cycle arrest in S-phase. However, the pharmacological target of PUB-SOs was still unidentified. Consequently, the objective of the present study was to identify and confirm the pharmacological target of the prototypical PUB-SO named 2-ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) leading to the cell cycle arrest in S-phase. The antiproliferative and the cytotoxic activities of SFOM-0046 were characterized using the NCI-60 screening program and its fingerprint was analyzed by COMPARE algorithm. Then, human dihydroorotate dehydrogenase (hDHODH) colorimetric assay, uridine rescuing cell proliferation and molecular docking in the brequinar-binding site were performed. As a result, SFOM-0046 exhibited a mean antiproliferative activity of 3.5 μM in the NCI-60 screening program and evidenced that leukemia and colon cancer cell panels were more sensitive to SFOM-0046. COMPARE algorithm showed that the SFOM-0046 cytotoxic profile is equivalent to the ones of brequinar and dichloroallyl lawsone, two inhibitors of hDHODH. SFOM-0046 inhibited the hDHODH in the low nanomolar range (IC₅₀ = 72 nM) and uridine rescued the cell proliferation of HT-29, HT-1080, M21 and MCF-7 cancer cell lines in the presence of SFOM-0046. Finally, molecular docking showed a binding pose of SFOM-0046 interacting with Met43 and Phe62 present in the brequinar-binding site. In conclusion, PUB-SOs and notably SFOM-0046 are new small molecules hDHODH inhibitors triggering replication stresses and S-phase arrest.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Where all the Roads Meet? A Crossover Perspective on Host Factors Regulating SARS-CoV-2 infection

COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.     

Call for biotechnological approach to degrade plastic in the era of COVID-19 pandemic

Plastic pollution is a global issue and has become a major concern since Coronavirus disease (COVID)-19. In developing nations, landfilling and illegal waste disposal are typical ways to dispose of COVID-19-infected material. These technologies worsen plastic pollution and other human and animal health problems. Plastic degrades in light and heat, generating hazardous primary and secondary micro-plastic. Certain bacteria can degrade artificial polymers using genes, enzymes, and metabolic pathways. Microorganisms including bacteria degrade petrochemical plastics slowly. High molecular weight, strong chemical bonds, and excessive hydrophobicity reduce plastic biodegradation. There is not enough study on genes, enzymes, and bacteria-plastic interactions. Synthetic biology, metabolic engineering, and bioinformatics methods have been created to biodegrade synthetic polymers. This review will focus on how microorganisms' degrading capacity can be increased using recent biotechnological techniques.     

Ecological analysis and environmental niche modelling of Dactylorhiza hatagirea (D. Don) Soo: A conservation approach for critically endangered medicinal orchid

The natural populations of Dactylorhiza hatagirea have been greatly affected due to incessant exploitation. As such, studies on its population attributes together with habitat suitability and environmental factors affecting its distribution are needed to be undertaken for its conservation in nature. Present study aimed at accessing an impact of anthropogenic pressure on population structure and locate suitable habitats for the conservation of this critically endangered orchid. Considerable changes in the phytosociological attributes were observed on account of the changing magnitude and extent of anthropogenic threat in their natural abode. The distribution pattern of species indicated that more than 90% of the populations exhibit substantially aggregated spatial distribution. Maximum Entropy (MaxEnt) distribution modelling algorithm was used to predict suitable habitat and potential area for its cultivation and reintroduction. Twenty-seven occurrence records, nineteen bioclimatic variables, altitude, and slope were used. MaxEnt map output gave the habitat suitability for this species and predicted its distribution in the North-Western Himalayas of India for approximately 616 km². Jackknifing indicated that maximum temperature of warmest month, annual mean temperature, mean temperature of the driest quarter, and mean temperature of the wettest quarter were the governing factors for its distribution and hence, presented a higher gain with respect to other variables. According to permutation importance, precipitation seasonality and mean temperature of wettest quarter shows the prominent impact on the habitat distribution. Results of AUC (area under curve) were statistically significant (0.940) and the line of predicted omission falls very close to an omission on training samples, validating a better run of the model. Response curves revealed a probable increase in the occurrence of D. hatagirea with an increase in mean temperature of the wettest quarter and maximum temperature of the warmest month contributed more than 50% to predicted habitat suitability. Direct field observations concurrent with predicted habitat suitability and google-earth images represent greater model thresholds for successful inception of the species. Together, the study proposes that the species can be conserved in or near its present-day natural habitats and is equally effective in determining the possible habitats for its cultivation and reintroduction.     

Inhibition of Mycobacterium tuberculosis InhA: Design,synthesis and evaluation of new di-triclosan derivatives

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC₅₀ of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC₉₉ of 12.9 µM against M. bovis.     

Studies on fragment-based design of allosteric inhibitors of human factor XIa

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa’s active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.     

Meniscal lesion detection and characterization in adult knee MRI: A deep learning model approach with external validation

PurposeEvaluation of a deep learning approach for the detection of meniscal tears and their characterization (presence/absence of migrated meniscal fragment).MethodsA large annotated adult knee MRI database was built combining medical expertise of radiologists and data scientists’ tools. Coronal and sagittal proton density fat suppressed-weighted images of 11,353 knee MRI examinations (10,401 individual patients) paired with their standardized structured reports were retrospectively collected. After database curation, deep learning models were trained and validated on a subset of 8058 examinations. Algorithm performance was evaluated on a test set of 299 examinations reviewed by 5 musculoskeletal specialists and compared to general radiologists’ reports. External validation was performed using the publicly available MRNet database. Receiver Operating Characteristic (ROC) curves results and Area Under the Curve (AUC) values were obtained on internal and external databases.ResultsA combined architecture of meniscal localization and lesion classification 3D convolutional neural networks reached AUC values of 0.93 (95% CI 0.82, 0.95) for medial and 0.84 (95% CI 0.78, 0.89) for lateral meniscal tear detection, and 0.91 (95% CI 0.87, 0.94) for medial and 0.95 (95% CI 0.92, 0.97) for lateral meniscal tear migration detection. External validation of the combined medial and lateral meniscal tear detection models resulted in an AUC of 0.83 (95% CI 0.75, 0.90) without further training and 0.89 (95% CI 0.82, 0.95) with fine tuning.ConclusionOur deep learning algorithm demonstrated high performance in knee menisci lesion detection and characterization, validated on an external database.     

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)₂, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.     

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral M^pro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of M^pro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 M^pro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the M^pro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.     

Turbinaria ornata and its associated epiphytic Bacillus sp. A promising molecule supplier to discover new natural product approaches

Marine ecosystems are highly dependent on macroalgea in providing food and shelter for aquatic organisms, interacting with many bacteria and mostly producing secondary metabolites of potent therapeutic antibacterial property. Screening of marine microbial secondary metabolites of valuable biotechnological and therapeutical applications are now extensively studied. In this study, Bacillus spp. identified by DNA sequencing and found associated with Turbinaria ornata, was screened and characterized for its cell free supernatant (CFS) possible antimicrobial and antibiofilm applications. Among the 7 microbial isolates tested, CFS greatly affected Bacillus subitilis (12 mm) and inhibited equally the yeast isolates Candida albicans, Candida tropicalis and Candida glabrata (10 mm) and had no or negligible effect on S.aureus, E.coli, P. aeruginosa. As for the CFS antibiofilm activity, no difference was revealed from the positive control. Algal crude extracts (methanol, acetone and aqueous), on the other hand, were similarly tested for their antimicrobial activity against the seven microbial isolates, where highest activity was observed with the aqueous crude extract against Staphylococcus aureus(10 mm) and Pseudomonas aeruginosa (9 mm) compared to the negligible effects of methanol and acetone crude extracts. Chemical analysis was performed to reveal the major constituents of both crude algal extracts and Bacillus spp. CFS. FTIR spectrum of the bacterial CFS indicated the presence of bacteriocin as the major lipopeptide responsible for its biological activity. Whereas, methanol and water crude algal extract GC–MS spectra revealed different chemical groups of various combined therapeutical activity mainly Naphthalene, amino ethane-sulfonic acid, pyrlene, Biotin and mercury chloromethyl correspondingly. Thus, the present study, demonstrated the moderate activity of both crude algal extract and the bacterial CFS, however, further investigations are needed for a better biological activity.     

Discovery and structure activity relationships of 7-benzyl triazolopyridines as stable,selective, and reversible inhibitors of myeloperoxidase

Myeloperoxidase (MPO) is a heme peroxidase found in neutrophils, monocytes and macrophages that efficiently catalyzes the oxidation of endogenous chloride into hypochlorous acid for antimicrobial activity. Chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. Triazolopyrimidine 5 is a reversible MPO inhibitor; however it suffers from poor stability in acid, and is an irreversible inhibitor of the DNA repair protein methyl guanine methyl transferase (MGMT). Structure-based drug design was employed to discover benzyl triazolopyridines with improved MPO potency, as well as acid stability, no reactivity with MGMT, and selectivity against thyroid peroxidase (TPO). Structure-activity relationships, a crystal structure of the MPO-inhibitor complex, and acute in vivo pharmacodynamic data are described herein.     

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25–25 µg/ml) for an hour then incubated with H₂O₂ (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H₂O₂. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H₂O₂ only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.