Secondary metabolites produced by endophytic fungi, Alternaria alternata, as potential inhibitors of the human immunodeficiency virus

Antiretroviral treatment has significantly reduced human immunodeficiency virus infection and mortality. However, the current treatment regimen is limited by adverse side effects, the emergence of drug resistance, and the inability to eliminate viral reservoirs. Here, fifteen endophytic fungi were isolated from Sclerocarya birrea and Hypoxis plants. Crude extracts of Alternaria alternata (strain ID PO4PR1, PO4PR2, and PO2PL1) of the fifteen isolate’s crude extracts showed anti-HIV-1 activity in TZM-bl cell line at inhibitory concentration (IC50) values ranging from 0.017 to 1.170 μg/ml. The three crude extracts also maintained the virus replication inhibition profile on PBMCs and CD4+ T cells at concentrations ranging from 0.3 to 50.2 ng/ml. Partial purification using the solid phase extraction and analysis with Gas Chromatography-Mass spectrophotometry showed a diverse profile. The bioactive compounds were identified based on peak area, retention time, similarity index. The major compounds from GC-MS analysis of A. Alternata revealed the existence of cyclotrisiloxane octamethyl (22.92%); Propaninitrile (16,67%); Pyrrolol[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl) (10.42%); Silane, diethylethoxy(2-ethoxyethyloxy) (4.17%); Coumarin, 3,4-dihydro-4,5,7-trimethyl- 4,5,7-Trimethyl-2-chromanone (13.7%) and 1,2-Cyclobutanedicarbonitrile (2.08%) with previously reported biological activities such as antimicrobial, anti-inflammatory and antioxidant properties. Therefore, these bioactive compounds from A. alternata fungal endophytes could be repurposed as potential anti-HIV agents. This study showed the potential of endophytic fungi, Alternaria alternata from S. birrea, and Hypoxis species as producers of anti-HIV compounds

1,4,7-Triazacyclononane restores the activity of β-lactam antibiotics against metallo-β-lactamase-producing Enterobacteriaceae : exploration of potential metallo-β-lactamase inhibitors

Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae. MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min−1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.The South African National Research Foundation (grant no. 85595 awarded to S. Y. Essack as incentive funding for rated researchers) and the College of Health Sciences, University of Kwa-Zulu Natal.http://aem.asm.org2019-08-01hj2019Medical Microbiolog

In Silico Drug Repurposing of FDA-Approved Drugs Highlighting Promacta as a Potential Inhibitor of H7N9 Influenza Virus

Influenza virus infections continue to be a significant and recurrent public health problem. Although vaccine efficacy varies, regular immunisation is the most effective method for suppressing the influenza virus. Antiviral drugs are available for influenza, although two of the four FDA-approved antiviral treatments have resulted in significant drug resistance. Therefore, new treatments are being sought to reduce the burden of flu-related illness. The time-consuming development of treatments for new and re-emerging diseases such as influenza and the high failure rate are increasing concerns. In this context, we used an in silico-based drug repurposing method to repurpose FDA-approved drugs as potential therapies against the H7N9 virus. To find potential inhibitors, a total of 2568 drugs were screened. Promacta, tucatinib, and lurasidone were identified as promising hits in the DrugBank database. According to the calculations of MM-GBSA, tucatinib (−54.11 kcal/mol) and Promacta (−56.20 kcal/mol) occupied the active site of neuraminidase with a higher binding affinity than the standard drug peramivir (−49.09 kcal/mol). Molecular dynamics (MD) simulation studies showed that the C-α atom backbones of the complexes of tucatinib and Promacta neuraminidase were stable throughout the simulation period. According to ADME analysis, the hit compounds have a high gastrointestinal absorption (GI) and do not exhibit properties that allow them to cross the blood–brain barrier (BBB). According to the in silico toxicity prediction, Promacta is not cardiotoxic, while lurasidone and tucatinib show only weak inhibition. Therefore, we propose to test these compounds experimentally against the influenza H7N9 virus. The investigation and validation of these potential H7N9 inhibitors would be beneficial in order to bring these compounds into clinical settings

Impact of the R292K Mutation on Influenza A (H7N9) Virus Resistance towards Peramivir: A Molecular Dynamics Perspective

In March 2013, a novel avian influenza A (H7N9) virus emerged in China. By March 2021, it had infected more than 1500 people, raising concerns regarding its epidemic potential. Similar to the highly pathogenic H5N1 virus, the H7N9 virus causes severe pneumonia and acute respiratory distress syndrome in most patients. Moreover, genetic analysis showed that this avian H7N9 virus carries human adaptation markers in the hemagglutinin and polymerase basic 2 (PB2) genes associated with cross-species transmissibility. Clinical studies showed that a single mutation, neuraminidase (NA) R292K (N2 numbering), induces resistance to peramivir in the highly pathogenic H7N9 influenza A viruses. Therefore, to evaluate the risk for human public health and understand the possible source of drug resistance, we assessed the impact of the NA-R292K mutation on avian H7N9 virus resistance towards peramivir using various molecular dynamics approaches. We observed that the single point mutation led to a distorted peramivir orientation in the enzyme active site which, in turn, perturbed the inhibitor’s binding. The R292K mutation induced a decrease in the interaction among neighboring amino acid residues when compared to its wild-type counterpart, as shown by the high degree of fluctuations in the radius of gyration. MM/GBSA calculations revealed that the mutation caused a decrease in the drug binding affinity by 17.28 kcal/mol when compared to the that for the wild-type enzyme. The mutation caused a distortion of hydrogen bond-mediated interactions with peramivir and increased the accessibility of water molecules around the K292 mutated residue

Investigation of flap flexibility of β-secretase using molecular dynamic simulations

<p>Flap motif and its dynamics were extensively reported in aspartate proteases, e.g. HIV proteases and plasmepsins. Herein, we report the first account of flap dynamics amongst different conformations of β-secretase using molecular dynamics simulation. Various parameters were proposed and a selected few were picked which could appropriately describe the flap motion. Three systems were studied, namely Free (BACE_Free) and two ligand-bound conformations, which belonged to space groups P6₁22 (BACE_Bound1) and C222₁ (BACE_Bound2), respectively and four parameters (distance between the flaps tip residue, Thr72 and Ser325, <i>d</i>₁; dihedral angle, <i>ϕ</i> (Thr72-Asp32-Asp228-Ser325); TriCα angles, <i>θ</i>₁ (Thr72-Asp32-Ser325), and <i>θ</i>₂ (Thr72-Asp228-Ser325)) were proposed to understand the change in dynamics of flap domain and the extent of flap opening and closing. Analysis of, <i>θ</i>₂, <i>d</i>₁, <i>θ</i>₁ and <i>ϕ</i> confirmed that the BACE_Free adopted semi-open, open and closed conformations with slight twisting during flap opening. However, BACE_Bound1 (P6122) showed an adaptation to open conformation due to lack of hydrogen bond interaction between the ligand and flap tip residue. A slight flap <i>twisting, ϕ</i> (<i>lateral twisting</i>) was observed for BACE_Bound1 during flap opening which correlates with the opening of BACE_Free. Contradictory to the BACE_Bound1, the BACE_Bound2 locked the flap in a closed conformation throughout the simulation due to formation of a stable hydrogen bond interaction between the flap tip residue and ligand. Analyses of all three systems highlight that <i>d</i>₁, <i>θ</i>₂ and <i>ϕ</i> can be precisely used to describe the extent of flap opening and closing concurrently with snapshots along the molecular dynamics trajectory across several conformations of β-secretase.</p

A Comprehensive Analysis of Structural and Functional Changes Induced by SARS-CoV-2 Spike Protein Mutations

The emergence of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has sparked intense research on its spike protein, which is essential for viral entrance into host cells. Viral reproduction and transmission, host immune response regulation, receptor recognition and host cell entrance mechanisms, as well as structural and functional effects have all been linked to mutations in the spike protein. Spike protein mutations can also result in immune evasion mechanisms that impair vaccine effectiveness and escape, and they are linked to illness severity and clinical consequences. Numerous studies have been conducted to determine the effects of these mutations on the spike protein structure and how it interacts with host factors. These results have important implications for the design and development of medicines and vaccines based on spike proteins as well as for the assessment of those products’ efficiency against newly discovered spike protein mutations. This paper gives a general overview of how spike protein mutations are categorized and named. It further looks at the links between spike protein mutations and clinical outcomes, illness severity, unanswered problems, and future research prospects. Additionally, explored are the effects of these mutations on vaccine effectiveness as well as the possible therapeutic targeting of spike protein mutations

Allostery Inhibition of BACE1 by Psychotic and Meroterpenoid Drugs in Alzheimer’s Disease Therapy

In over a century since its discovery, Alzheimer’s disease (AD) has continued to be a global health concern due to its incurable nature and overwhelming increase among older people. In this paper, we give an overview of the efforts of researchers towards identifying potent BACE1 exosite-binding antibodies and allosteric inhibitors. Herein, we apply computer-aided drug design (CADD) methods to unravel the interactions of some proposed psychotic and meroterpenoid BACE1 allosteric site inhibitors. This study is aimed at validating the allosteric potentials of these selected compounds targeted at BACE1 inhibition. Molecular docking, molecular dynamic (MD) simulations, and post-MD analyses are carried out on these selected compounds, which have been experimentally proven to exhibit allosteric inhibition on BACE1. The SwissDock software enabled us to identify more than five druggable pockets on the BACE1 structural surface using docking. Besides the active site region, a melatonin derivative (compound 1) previously proposed as a BACE1 allostery inhibitor showed appreciable stability at eight different subsites on BACE1. Refinement with molecular dynamic (MD) simulations shows that the identified non-catalytic sites are potential allostery sites for compound 1. The allostery and binding mechanism of the selected potent inhibitors show that the smaller the molecule, the easier the attachment to several enzyme regions. This finding hereby establishes that most of these selected compounds failed to exhibit strong allosteric binding with BACE1 except for compound 1. We hereby suggest that further studies and additional identification/validation of other BACE1 allosteric compounds be done. Furthermore, this additional allosteric site investigation will help in reducing the associated challenges with designing BACE1 inhibitors while exploring the opportunities in the design of allosteric BACE1 inhibitors

Prevalence and Antimicrobial Resistance of Escherichia coli Isolated from Various Meat Types in the Tamale Metropolis of Ghana

Meats are important potential sources of foodborne pathogens including Escherichia coli. This study was conducted to determine the prevalence and antimicrobial resistance of Escherichia coli isolated from meats in the Tamale metropolis of Ghana. Isolation of Escherichia coli was done using the procedure according to the USA-FDA Bacteriological Analytical Manual. Antibiotic resistance patterns in the Escherichia coli isolates were determined by the Kirby-Bauer disk diffusion method against 8 antibiotics. The overall prevalence of Escherichia coli in the meat samples was 84.00% (189/225). Mutton (88.89%), guinea fowl (88.89%), beef (86.67%), local chicken (80.00%), and chevon (75.56%) were contaminated by Escherichia coli. The average coliform count was 4.22 cfu/cm2 and was highest in guinea fowl (4.94 log cfu/cm2) and lowest in local chicken (3.23 log cfu/cm2). The Escherichia coli isolates were highly resistant to erythromycin (85.00%), tetracycline (73.33%), and ampicillin (71.67%). The multiple antibiotic resistance (MAR) index ranged from 0.13 to 1. The Escherichia coli isolates exhibited 23 antimicrobial resistance patterns with resistant pattern TeAmpE (tetracycline-ampicillin-erythromycin) being the most common. Multidrug resistance was 68.33% (41/60) among the Escherichia coli isolates. The results showed that Escherichia coli was commonly present in the various meat types and exhibited multidrug resistances, necessitating efficient antibiotic stewardship guidelines to streamline their use in the production industry

1,4,7-Triazacyclononane Restores the Activity of β-Lactam Antibiotics against Metallo-β-Lactamase-Producing Enterobacteriaceae

Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae. MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min−1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.The South African National Research Foundation (grant no. 85595 awarded to S. Y. Essack as incentive funding for rated researchers) and the College of Health Sciences, University of Kwa-Zulu Natal.http://aem.asm.org2019-08-01hj2019Medical Microbiolog

Intermolecular Mechanism and Dynamic Investigation of Avian Influenza H7N9 Virus&rsquo; Susceptibility to E119V-Substituted Peramivir&ndash;Neuraminidase Complex

The H7N9 virus attaches itself to the human cell receptor protein containing the polysaccharide that terminates with sialic acid. The mutation of neuraminidase at residue E119 has been explored experimentally. However, there is no adequate information on the substitution with E119V in peramivir at the intermolecular level. Therefore, a good knowledge of the interatomic interactions is a prerequisite in understanding its transmission mode and subsequent effective inhibitions of the sialic acid receptor cleavage by neuraminidase. Herein, we investigated the mechanism and dynamism on the susceptibility of the E119V mutation on the peramivir&ndash;neuraminidase complex relative to the wildtype complex at the intermolecular level. This study aims to investigate the impact of the 119V substitution on the neuraminidase&ndash;peramivir complex and unveil the residues responsible for the complex conformations. We employed molecular dynamic (MD) simulations and extensive post-MD analyses in the study. These extensive computational investigations were carried out on the wildtype and the E119V mutant complex of the protein for holistic insights in unveiling the effects of this mutation on the binding affinity and the conformational terrain of peramivir&ndash;neuraminidase E119V mutation. The calculated total binding energy (&Delta;Gbind) for the peramivir wildtype is &minus;49.09 &plusmn; 0.13 kcal/mol, while the E119V mutant is &minus;58.55 &plusmn; 0.15 kcal/mol. The increase in binding energy (9.46 kcal/mol) is consistent with other post-MD analyses results, confirming that E119V substitution confers a higher degree of stability on the protein complex. This study promises to proffer contributory insight and additional knowledge that would enhance future drug designs and help in the fight targeted at controlling the avian influenza H7N9 virus. Therefore, we suggest that experimentalists collaborate with computational chemists for all investigations of this topic, as we have done in our previous studies