In silico identification of chikungunya virus replication inhibitor validated using biochemical and cell-based approaches.

Discovering an alternative therapy with a long-lasting effect on symptoms caused by chikungunya virus (CHIKV) infection is prompted by the lack of a vaccine and the absence of safe, effective and non-toxic medications. One potential strategy is synthesizing or identifying small compounds that can specifically target the active site of an essential enzyme and prevent virus replication. Previous site-directed mutagenesis studies have demonstrated the crucial role of the macrodomain, which is a part of non-structural protein 3 (nsP3), in virus replication. Exploiting this fact, the macrodomain can be targeted to discover a natural substance that can inhibit its function and thereby impede virus replication. With this aim, the present study focused on potential CHIKV nsP3 macrodomain (nsP3MD) inhibitors through in silico, in vitro and cell-based methods. Through virtual screening of the natural compound library, nine nsP3MD inhibitors were initially identified. Molecular dynamics (MD) simulations were employed to evaluate these nine compounds based on the stability of their ligand-receptor complexes and energy parameters. Target analysis and ADMET (i.e. absorption, distribution, metabolism, excretion and toxicity) prediction of the selected compounds revealed their drug-like characteristics. Subsequent in vitro investigation allowed us to narrow the selection down to one compound, N-[2-(5-methoxy-1H-indol-3-yl) ethyl]-2-oxo-1,2-dihydroquinoline-4-carboxamide, which exhibited potent inhibition of CHIKV growth. This molecule effectively inhibited CHIKV replication in the stable embryonal rhabdomyosarcoma cell line capable of producing CHIKV. Our findings demonstrate that the selected compound possesses substantial anti-CHIKV nsP3MD activity both in vitro and in vivo. This work provides a promising molecule for further preclinical studies to develop a potential drug against the CHIKV.

Molecular docking and dynamic simulation analysis of Hepatitis E virus protease in complexing with the E64 inhibitor.

The unavailability of a suitable treatment for human Hepatitis E virus (HEV) infection necessitate the development of anti HEV drugs. The HEV papain-like cysteine proteases (HEV PCP) is a crucial target to prevent viral replication and progression. E64 is a known HEV PCP inhibitor; however, its molecular mechanism of inhibition is not yet known. Since the crystal structure of HEV PCP is not available, the primary focuses of the present study was to refine the predicted HEV PCP structural model by molecular dynamics (MD) simulation. Further, we performed a 200 ns MD simulation to understand the structural complexity of HEV PCP and the effect of E64 binding with HEV PCP. The E64 binding with active site residues Gln48, Thr51, Gln55, Cys52, Ser81, Gln 98, Cys 132, Arg158, His159, Asn 160 and Ala96 leads to reduced fluctuations in the residue at N-terminal (18-41) that include the CHC motif (26-28). However, most of the other non interacting residues, including the inter-domain linker region (46-87), showed increased fluctuations in the HEV PCP-E64 complex. The residue Asp21 and Ala96 are involved in the formation of interdomain interactions in the HEV PCP apo enzyme. While in the PCP-E64 complex, E64 binds to Ala96 and creates a steric hindrance to prevent interdomain interactions. Thus, the E64 binding reduces interdomain interactions and restrict domain movements in the HEV PCP-E64 complex. This information will be important for the chemically designing more effective derivatives of E64 developing HEV PCP specific inhibitors.Communicated by Ramaswamy H. Sarma.

Inhibition of Hepatitis E Virus Replication by Novel Inhibitor Targeting Methyltransferase.

Hepatitis E Virus (HEV) is a quasi-enveloped virus having a single-stranded, positive-sense RNA genome (~7.2 kb), flanked with a 5' methylated cap and a 3' polyadenylated tail. The HEV open reading frame 1 (ORF1) encodes a 186-kDa polyprotein speculated to get processed and produce Methyltransferase (MTase), one of the four essential replication enzymes. In this study, we report the identification of the MTase inhibitor, which may potentially deplete its enzymatic activity, thus causing the cessation of viral replication. Using in silico screening through docking, we identified ten putative compounds, which were tested for their anti-MTase activity. This resulted in the identification of 3-(4-Hydroxyphenyl)propionic acid (HPPA), with an IC50 value of 0.932 ± 0.15 µM, which could be perceived as an effective HEV inhibitor. Furthermore, the compound was tested for inhibition of HEV replication in the HEV culture system. The viral RNA copies were markedly decreased from ~3.2 × 106 in untreated cells to ~4.3 × 102.8 copies in 800 µM HPPA treated cells. Therefore, we propose HPPA as a potential drug-like inhibitor against HEV-MTase, which would need further validation through in vivo analysis using animal models and the administration of Pharmacokinetic and Pharmacodynamic (PK/PD) studies.

In silico identification and validation of natural antiviral compounds as potential inhibitors of SARS-CoV-2 methyltransferase.

The novel Coronavirus disease 2019 (COVID-19) is potentially fatal and caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Due to the unavailability of any proven treatment or vaccination, the outbreak of COVID-19 is wreaking havoc worldwide. Hence, there is an urgent need for therapeutics targeting SARS-CoV-2. Since, botanicals are an important resource for several efficacious antiviral agents, natural compounds gaining significant attention for COVID-19 treatment. In the present study, methyltranferase (MTase) of the SARS-CoV-2 is targeted using computational approach. The compounds were identified using molecular docking, virtual screening and molecular dynamics simulation studies. The binding mechanism of each compound was analyzed considering the stability and energetic parameter using in silico methods. We have found four natural antiviral compounds Amentoflavone, Baicalin, Daidzin and Luteoloside as strong inhibitors of methyltranferase of SARS-CoV-2. ADMET prediction and target analysis of the selected compounds showed favorable results. MD simulation was performed for four top-scored molecules to analyze the stability, binding mechanism and energy requirements. MD simulation studies indicated energetically favorable complex formation between MTase and the selected antiviral compounds. Furthermore, the structural effects on these substitutions were analyzed using the principles of each trajectories, which validated the interaction studies. Our analysis suggests that there is a very high probability that these compounds may have a good potential to inhibit Methyltransferase (MTase) of SARS-CoV-2 and to be used in the treatment of COVID-19. Further studies on these natural compounds may offer a quick therapeutic choice to treat COVID-19.Communicated by Ramaswamy H. Sarma.

In silico identification of natural antiviral compounds as a potential inhibitor of chikungunya virus non-structural protein 3 macrodomain.

Chikungunya Virus (CHIKV) is having a major impact on humans with potentially life-threatening and debilitating arthritis. The lack of a specific antiviral drug against the CHIKV disease has created an alarming situation to identify or develop potent chemical molecules for its remedial measures. Antiviral therapies for viral diseases are generally expensive and have adverse side effects. Plant-based antiviral natural compounds are the most suitable and best alternative of current antiviral drugs because of less toxicity. In the present study, non-structural protein 3 macrodomain (nsP3MD) of the CHIKV that is essential for virus replication has been selected for anti CHIKV drug target. The compounds were identified using molecular docking, virtual screening and further evaluated by molecular dynamics (MD) simulation studies. The binding mechanism of each compound was analyzed considering the stability and energetic parameter. We have found six plant-based natural antiviral compounds Baicalin, Rutaecarpine, Amentoflavone, Apigetrin, Luteoloside, and Baloxavir as strong inhibitors of nsP3MD of CHIKV. ADMET prediction and target analysis of the selected compounds showed drug likeliness of these compounds. MD simulation studies indicated energetically favorable complex formation between nsP3MD and the selected antiviral compounds. Furthermore, the structural effects on these substitutions were analyzed using the principles of each trajectory, which validated the interaction studies. Our analysis suggests a very high probability of these compounds to inhibit nsP3MD of CHIKV and could be evaluated for Chikungunya fever drug development. Communicated by Ramaswamy H. Sarma.

In silico identification and evaluation of Bacillus subtilis cold shock protein B (cspB)-like plant RNA chaperones.

Cold shock domain (CSD) proteins with nucleic acid binding properties are well conserved from bacteria to higher organisms. In bacteria, the cold shock proteins (CSPs) are single domain RNA chaperones, whereas in animals and plants, CSDs are accompanied by additional domains with roles in transcription regulation. Bacterial CSPs (Escherischia coli-cspA and Bacilus subtilis-cspB) have successfully imparted drought tolerance in transgenic plants; however, these cannot be deployed in food crops due to their low public acceptance of transgenics with bacterial genes. Therefore, this study aimed to identify CSPB-like proteins from plants that can be used for developing drought tolerant transgenic crops. Twelve single domain plant CSPs presenting >40% sequence identity with CSPB were identified. All 12 plant CSPs were modeled by homology modeling and refined by molecular dynamics simulation for 10 ns. Selected plant CSPs and CSPB exhibited high structural similarity (Tm-score: 0.63-0.86). Structure based phylogenetic analysis revealed that Triticum aestivum-csp1 and Aegilops tauschii-cspE are structurally closer to CSPB compared to their orthologs and paralogs. Molecular docking with three RNA molecules (5U, UC3U, and C2UC) indicates that Ricinus communis-csd1 and T. aestivum-csp1 have a binding pattern and docking scores similar to those of CSPB. Furthermore, MD simulations for 20 ns and analysis of RMSD, RMSF, Rg as well as the number of hydrogen bonds in all the three complexes revealed that plant CSP-RNA complexes behave in a similar manner to that of the CSPB-RNA complex, making them highly potential candidate genes for developing drought tolerance in transgenic plants. Communicated by Ramaswamy H. Sarma.

Baseline characterization of sediments and marine biota near industrial effluent discharge in Northumberland Strait, Nova Scotia, Canada.

A bleached kraft pulp mill operating in Nova Scotia, Canada has discharged effluent into a former tidal estuary known as Boat Harbour since 1967. After treatment in Boat Harbour, effluent is discharged into Northumberland Strait. Contaminated sediments in Boat Harbour are slated for remediation following cessation of effluent discharge. A review of historical documents to identify contaminants in marine biota in Northumberland Strait found insufficient data to properly assess baseline conditions prior to remediation. This study measured metal, methylmercury, dioxin and furan concentrations in surficial sediments and American lobster (Homarus americanus), rock crabs (Cancer irroratus) and blue mussels (Mytilus edulis) in Northumberland Strait. When compared to Canadian Council of Ministers of the Environment sediment quality guidelines and Canadian Food Inspection Agency tissue guidelines results indicated limited contamination in sediments and biota, posing low risk to marine biota. Long-term monitoring is recommended to verify effectiveness of remediation.

Baseline assessment of contaminants in marine biota prior to remediation of industrial effluent impacted sediments in a former tidal estuary in Nova Scotia, Canada.

Contaminated sediments at a pulp mill and former chor-alkali effluent treatment facility in Nova Scotia, Canada will undergo remediation. However, baseline studies assessing contaminants in marine biota in the marine receiving environment are lacking. Historical qualitative and quantitative contaminant data in biota from Boat Harbour (a former tidal lagoon which was used to treat industrial effluent since 1967), and surrounding marine environment were reviewed to establish baseline pollution from industrial effluent and contaminated sediments. Elevated metal, dioxins and furan concentrations previously measured in marine biota needs updating to help inform pre-remediation monitoring. Selection of species, contaminants of concern and sampling locations were ad hoc and often inconsistent with environmental effects monitoring requirements under Canadian federal Pulp and Paper Effluent Regulations. These consolidated baseline data are required to determine historical impacts and to assist future monitoring during Boat Harbour sediment remediation to compare against.

Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays.

Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432-592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in E. coli and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through in silico analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of r2 = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of "Papain-like cysteine protease," as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established.

Reducing marine pollution from single-use plastics (SUPs): A review.

Single-use plastics, or SUPs (plastic bags, microbeads, cutlery, straws and polystyrene) are substantial sources of plastic marine pollution, yet preventable via legislative and non-legislative interventions. Various international legislative strategies have been reported to address plastic marine pollution from plastic bags and microbeads, but these have since been accompanied by recent increasing public awareness triggered by international agencies and organizations. The Sixth International Marine Debris Conference highlighted increasing intervention strategies to mitigate SUP pollution. This study presents new multi-jurisdictional legislative interventions to reduce SUPs since 2017 and incorporates emergence of new non-legislative interventions to mitigate other types of SUPs at individual and private-sector levels that complement or influence legislative interventions. Further, effectiveness of SUP bag interventions (e.g., bans vs. levies) to help reduce SUP marine pollution are presented and range between 33 and 96% reduction in bag use.

Screening and In Vitro Evaluation of Potential Plasmodium falciparum Leucyl Aminopeptidase Inhibitors.

BACKGROUND: Plasmodium falciparum leucyl aminopeptidase (PfA-M17) regulates the intracellular pool of amino acids required for the growth and development of parasites. Thus, PfA-M17 is a promising target for anti-malarial drug development. METHOD: In the present study, structure-based drug design was used to identify novel PfA-M17 inhibitors, which were subsequently validated by in vitro PfA-M17 and human LAP3 enzyme inhibition assay. A library of 3,147,882 compounds was screened using receptor-based virtual screening against the active site of PfA-M17, and three levels of accuracy were used: high-throughput virtual screening, gridbased ligand docking with energetics (Glide standard precision) and Glide extra precision. RESULTS: Seventeen screened compounds were selected and tested in the rPfA-M17 enzyme inhibition assay. Of these nine compounds were found to be effective inhibitors. To test the target activity, all nine PfA-M17 inhibitors were tested against rhLAP3, the human homolog of PfA-M17. One compound (compound 2) was found to be moderately effective against PfA-M17 (Ki = 287 µM) with limited inhibitory activity against hLAP3 (Ki of 4,464 µM). Subsequently, induced fit docking and pharmacophore modelling were used to further understand more precise ligand-protein interactions in the protein-inhibitor complexes. CONCLUSION: Among the 9 effective PfA-M17 inhibitors, 5 compounds were found effective in the P. falciparum schizont maturation inhibition (SMI) assay. A good correlation (r =0.83) was observed between the rPfA-M17 enzyme inhibition concentration and SMI assay.

Oral Manifestations and Molecular Basis of Oral Genodermatoses: A Review.

Genodermatoses refers to group of inherited monogenic disorders with skin manifestations. Many of these disorders are rare and also have oral manifestations, called oral genodermatoses. This article provides a focused review of molecular basis of important genodermatoses that affects the oral cavity and also have prominent associated dermatologic features. In several conditions discussed here, the oral findings are distinct and may provide the first clue of an underlying genetic diagnosis. The article also emphasises on the prenatal diagnosis, genetic counselling and the treatment oral genodermatoses.

Modelling of human leucyl aminopeptidases for in silico off target binding analysis of potential Plasmodium falciparum leucine aminopeptidase (PfA-M17) specific inhibitors.

Malaria is one of the most widespread infectious diseases in the world. Emergence of multi-drug resistant Plasmodium strains makes it crucial to identify new classes of compounds for anti-malarial therapy. Novel anti-malarial compounds from natural sources (Gomphostema niveum) as well as synthetic chemicals (5-aminolevulinic acid) have been reported in recent patents. Plasmodium falciparum leucyl aminopeptidase (PfA-M17) is a validated target for antimalarial drug development. However, known aminopeptidase inhibitors beset with the problem of non-specificity. Therefore, 3D structural models of PfA-M17 human homologs, Leucine aminopeptidase3 (hLAP3) and probable leucine aminopeptidase (hNPEPL1) were predicted for molecular docking based screening of potential inhibitors for their off target activity. Comparison of IC50 and docking scores of highly active hLAP3 inhibitors shows good correlation (r(2)≈ 0.8). Further, docking analysis with potential PfA-M17 inhibitor Compound-X (identified through virtual screening) shows much higher binding affinity towards PfA-M17 (docking score -11.44) than hLAP3 (docking score -4.26) and hNPEPL1 (docking score -5.08). This lead compound, Compound-X can act as a scaffold for further increasing PfA-M17 binding affinity and hLAP3 and hNPEPL1 3D structure models will be useful for screening of PfA-M17 specific inhibitors.

Modeling of human M1 aminopeptidases for in silico screening of potential Plasmodium falciparum alanine aminopeptidase (PfA-M1) specific inhibitors.

Plasmodium falciparum alanine M1-aminopeptidase (PfA-M1) is a validated target for anti-malarial drug development. Presence of significant similarity between PfA-M1 and human M1-aminopeptidases, particularly within regions of enzyme active site leads to problem of non-specificity and off-target binding for known aminopeptidase inhibitors. Molecular docking based in silico screening approach for off-target binding has high potential but requires 3D-structure of all human M1-aminopeptidaes. Therefore, in the present study 3D structural models of seven human M1-aminopeptidases were developed. The robustness of docking parameters and quality of predicted human M1-aminopeptidases structural models was evaluated by stereochemical analysis and docking of their respective known inhibitors. The docking scores were in agreement with the inhibitory concentrations elucidated in enzyme assays of respective inhibitor enzyme combinations (r2≈0.70). Further docking analysis of fifteen potential PfA-M1 inhibitors (virtual screening identified) showed that three compounds had less docking affinity for human M1-aminopeptidases as compared to PfA-M1. These three identified potential lead compounds can be validated with enzyme assays and used as a scaffold for designing of new compounds with increased specificity towards PfA-M1.