Targeting SARS-CoV-2 Nsp12/Nsp8 interactioninterface with approved and investigational drugs:an in silico structure-based approach

In this study, the Nsp12–Nsp8 complex of SARS-CoV-2 was targeted with structure-based and com-puter-aided drug design approach because of its vital role in viral replication. Sequence analysis ofRNA-dependent RNA polymerase (Nsp12) sequences from 30,366 different isolates were analysed forpossible mutations. FDA-approved and investigational drugs were screened for interaction with bothmutant and wild-type Nsp12–Nsp8 interfaces. Sequence analysis revealed that 70.42% of Nsp12sequences showed conserved P323L mutation, located in the Nsp8 binding cleft. Compounds werescreened for interface interaction, any with XP GScores lower than 7.0kcal/mol were considered aspossible interface inhibitors. RX-3117 (fluorocyclopentenyl cytosine) and Nebivolol had the highestbinding affinities in both mutant and wild-type enzymes, therefore they were selected and resultantprotein–ligand complexes were simulated for analysis of stability over 100ns. Although the selectedligands had partial mobility in the binding cavity, they were not removed from the binding pocketafter 100ns. The ligand RX-3117 remained in the same position in the binding pocket of the mutantand wild-type enzyme after 100ns MD simulation. However, the ligand Nebivolol folded andembedded in the binding pocket of mutant Nsp12 protein. Overall, FDA-approved and investigationaldrugs are able to bind to the Nsp12–Nsp8 interaction interface and prevent the formation of theNsp12–Nsp8 complex. Interruption of viral replication by drugs proposed in this study should be fur-ther tested to pave the way forin vivostudies towards the treatment of COVID-19

Targeting SARS-CoV-2 Nsp12/Nsp8 interaction interface with approved and investigational drugs: anin silicostructure-based approach

In this study, the Nsp12-Nsp8 complex of SARS-CoV-2 was targeted with structure-based and computer-aided drug design approach because of its vital role in viral replication. Sequence analysis of RNA-dependent RNA polymerase (Nsp12) sequences from 30,366 different isolates were analysed for possible mutations. FDA-approved and investigational drugs were screened for interaction with both mutant and wild-type Nsp12-Nsp8 interfaces. Sequence analysis revealed that 70.42% of Nsp12 sequences showed conserved P323L mutation, located in the Nsp8 binding cleft. Compounds were screened for interface interaction, any with XP GScores lower than -7.0 kcal/mol were considered as possible interface inhibitors. RX-3117 (fluorocyclopentenyl cytosine) and Nebivolol had the highest binding affinities in both mutant and wild-type enzymes, therefore they were selected and resultant protein-ligand complexes were simulated for analysis of stability over 100 ns. Although the selected ligands had partial mobility in the binding cavity, they were not removed from the binding pocket after 100 ns. The ligand RX-3117 remained in the same position in the binding pocket of the mutant and wild-type enzyme after 100 ns MD simulation. However, the ligand Nebivolol folded and embedded in the binding pocket of mutant Nsp12 protein. Overall, FDA-approved and investigational drugs are able to bind to the Nsp12-Nsp8 interaction interface and prevent the formation of the Nsp12-Nsp8 complex. Interruption of viral replication by drugs proposed in this study should be further tested to pave the way forin vivostudies towards the treatment of COVID-19

Evaluation of the potency of FDA-approved drugs on wild type and mutant SARS-CoV-2 helicase (Nsp13)

SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19

Introduction of novel thermostable alpha-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related alpha-amylases under five individual GH13 subfamilies

Among the thermophilic Bacillaceae family members, alpha-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus alpha-amylase genes displayed ae 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (ae 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (ae 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 alpha-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli alpha-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 alpha-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around alpha-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around alpha-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. (C) 2017 Elsevier Inc. All rights reserved

Cloning, expression and characterization of the gene encoding the enolase from Fusobacterium nucleatum

The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics. © 2016, Pleiades Publishing, Inc

Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System

One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein

Identification of benzamide inhibitors of histone deacetylase 1 from Babesia and Theileria species via high-throughput virtual screening and molecular dynamics simulations

Theileria and Babesia species are eukaryotic protozoan parasites classified under the order Piroplasmida of the phylum Apicomplexa. Tick vectors transmit these microorganisms in tropical and subtropical regions to a wide range of animals, including ruminants, causing fatal and life-threatening diseases such as bovine babesiosis and theileriosis. Resistance to commercially available drugs requires the search for new drug candidates. Histone deacetylase (HDAC) has a potential to be utilized as a drug target; therefore, it may be considered as an effective alternative. Previous studies revealed that HDAC inhibitors, identified for human use, show promising anti-parasitic effects. We have herein focused on the class I HDAC enzyme, HDAC1, of the Babesia and Theileria species to discover potential benzamide inhibitors by following a streamlined workflow of computer-aided drug design methodology. Molecular docking and molecular dynamics simulations revealed that benzamide derivatives stably interacted with the HDAC1 active site in both parasites as hypothesized. Furthermore, specific residue insertions at the entry point of the active site cleft of parasitic HDAC1 could enable ways to design parasite-specific drugs without adversely affecting host enzymes

Identification of potential inhibitors of Trichomonas vaginalis iron-containing superoxide dismutase by computer-aided drug design approach

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a common sexually transmitted disease. Metronidazole is a commonly used drug for the treatment of T. vaginalis infections. However, it was reported that the parasite has developed resistance to this drug. Therefore, a necessity of discovering new drugs that have different modes of action against T. vaginalis has emerged. In this computational study, T. vaginalis iron-containing superoxide dismutase (TvSOD) was selected as target protein because of its vital role in converting superoxide to oxygen and hydrogen peroxide and protecting the parasite against toxic reactive oxygen species (ROS). TvSOD was modeled using two different protein modeling programs, MODELLER and SWISS-MODEL, and then, small drug-like chemicals were screened for interaction with three different druggable pockets of the enzyme. The best interacting chemicals were then evaluated through molecular dynamics simulations (MDSs) for ligand stability. As a result, ligand-129817054 (7-(6-amino-1,2,3,4,5-pentahydroxyhexyl)-4-methylchromen-2-one) was determined to be a viable drug candidate based on docking scores and MDS results. Additional in vitro inhibition studies are necessary for the evaluation and assessment of the compound of interest as an effective TvSOD inhibitor