Ligand binding pocket of a novel Allatostatin receptor type C of stick insect, Carausius morosus

Allatostatins (AST) are neuropeptides with variable function ranging from regulation of developmental processes to the feeding behavior in insects. They exert their effects by binding to cognate GPCRs, called Allatostatin receptors (AlstR), which emerge as promising targets for pesticide design. However, AlstRs are rarely studied. This study is the first reported structural study on AlstR-AST interaction. In this work, the first C type AlstR from the stick insect Carausius morosus (CamAlstR-C) was identified and its interaction with type C AST peptide was shown to be physically consistent with the experimental results. The proposed structure of CamAlstR-C revealed a conserved motif within the third extracellular loop, which, together with the N-terminus is essential for ligand binding. In this work, computational studies were combined with molecular and nano-scale approaches in order to introduce an unknown GPCR-ligand system. Consequently, the data obtained provided a reliable target region for future agonist/inverse agonist studies on AlstRs

Screening of Clinically Approved and Investigation Drugs as Potential Inhibitors of SARS-CoV-2 Main Protease and Spike Receptor-Binding Domain Bound with ACE2 COVID19 Target Proteins: A Virtual Drug Repurposing Study

In this virtual drug repurposing study, we used 7922 FDA approved drugs and compounds in clinical investigation from NPC database. Both apo and holo forms of SARS-CoV-2 Main Protease as well as Spike Protein/ACE2 were used for virtual screening. Initially, docking was performed for these compounds at target binding sites. The compounds were then sorted according to their docking scores which represent binding energies. The first 100 compounds from each docking simulations were initially subjected to short (10 ns) MD simulations (in total 300 ligand-bound complexes), and average binding energies during MD simulations were calculated using the MM/GBSA method. Then, the selected promising hit compounds based on average MM/GBSA scores were used in long (100-ns and 500-ns) MD simulations. In total around 15 µs MD simulations were performed in this study. Both docking and MD simulations binding free energy calculations showed that holo form of the target protein is more appropriate choice for virtual drug screening studies. These numerical calculations have shown that the following 8 compounds can be considered as SARS-CoV-2 Main Protease inhibitors: Pimelautide, Rotigaptide, Telinavir, Ritonavir, Pinokalant, Terlakiren, Cefotiam and Cefpiramide. In addition, following 5 compounds were identified as potential SARS-CoV-2 ACE-2/Spike protein domain inhibitors: Denopamine, Bometolol, Naminterol, Rotigaptide and Benzquercin. These compounds can be clinically tested and if the simulation results validated, they may be considered to be used as treatment for COVID-19

Identifying and elucidating the roles of Y198N and Y204F mutations in the PAH enzyme through molecular dynamic simulations

Phenylketonuria is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase gene. In phenylketonuria causes various symptoms including severe mental retardation. PAH gene of a classical Phenylketonuria patient was sequenced, and two novel heterozygous mutations, p.Y198N and p.Y204F, were found. This study aimed to reveal the impacts of these variants on the structural stability of the PAH enzyme. In-silico analyses using prediction tools and molecular dynamics simulations were performed. Mutations were introduced to the wild type catalytic monomer and full length tetramer crystal structures. Variant pathogenicity analyses predicted p.Y198N to be damaging, and p.Y204F to be benign by some prediction tools and damaging by others. Simulations suggested p.Y198N mutation cause significant fluctuations in the spatial organization of two catalytic residues in the temperature accelerated MD simulations with the monomer and increased root-mean-square deviations in the tetramer structure. p.Y204F causes noticeable changes in the spatial positioning of T278 suggesting a possible segregation from the catalytic site in temperature accelerated MD simulations with the monomer. This mutation also leads to increased root-mean-square fluctuations in the regulatory domain which may lead to conformational change resulting in inhibition of dimerization and enzyme activation. Our study reports two novel mutations in the PAH gene and gives insight to their effects on the PAH activity. MD simulations did not yield conclusive results that explains the phenotype but gave plausible insight to possible effects which should be investigated further with in-silico and in-vitro studies to assess the roles of these mutations in etiology of PKU. Communicated by Ramaswamy H. Sarm

Lithium-mediated inhibition of GSK3β leads to β-catenin accumulation and increase in <i>MENA</i> mRNA levels.

<p>(A) Western blot analysis of Huh7 cells treated with either 25 mM NaCl or 25 mM LiCl for indicated timepoints. RT-PCR (B) and qRT-PCR (C) analysis of Huh7 cells treated with either 25 mM NaCl or 25 mM LiCl for indicated timepoints. (D) RT-PCR analysis of U373, Sk-Mel-103 and Sk-Mel-19 cells treated with LiCl for 24 hrs. Each image is a representative of at least two independent experiments.</p

Overexpression of β-catenin-S33Y and Wnt ligans leads to increase in <i>MENA</i> transcription.

<p>(A) β-catenin-S33Y mutant was stably overexpressed in Huh7 cells and <i>MENA</i> and Axin2 mRNA levels were analyzed using qRT-PCR. (B) β-catenin-S33Y was transiently overexpressed (48 hrs) in U373 cells and <i>MENA</i> and β-catenin mRNA levels were compared using RT-PCR. (C) Wnt1 or Wnt3a ligans were overexpressed in Huh7 cells (48 hrs) and <i>MENA</i> mRNA levels were compared using RT-PCR.</p

<i>Ena</i> knock-down induces tumor formation and metastasis <i>in vivo</i>.

<p><i>Ena</i> (A), <i>white</i> (B) and <i>axin</i> (C) knock-down was confirmed by RT-PCR. Actin was used as an internal control. Significant increase of tumor formation (D and E) and metastasis (F and G) in eyeful (Eyf) (D, F) and sensitized (St) (E, G) flies upon knock-down of <i>ena</i> and <i>axin</i> genes is observed, when <i>white</i> knock-down is used as a control. (* indicates p<0.05; *** indicates p<0.001 as analyzed by the chi-square test). Representative images of tumor and metastasis formations in eyeful and sensitized flies upon knock-down of <i>Ena</i> (H-O). (H and I) ey-GAL4, UAS-Dl, eyeful/+; UAS-EnaRNAi/+ (“+”: wild-type chromosome) flies showing tumors in the eye tissue. Every fold in the eye tissue is counted as a single tumor. (J and K) ey-Gal4, UAS-Dl/+, UAS-EnaRNAi/+ flies with tumors in the eye. (L and M) ey-GAL4, UAS-Dl, eyeful/+; UAS-EnaRNAi/+ flies showing metastasis on the abdomen and thorax. (N and O) ey-Gal4, UAS-Dl/+, UAS-EnaRNAi/+ flies with metastasis on the back and abdomen.</p

Promoters of mammalian <i>MENA</i> orthologs have functional Tcf4-binding elements (TBEs) that are regulated by the Wnt/β-catenin pathway.

<p>(A) Schematic representation of the promoters and the identified putative TBEs. The location of the TBEs is indicated by boxes and the sequences and locations with respect to transcriptional start sites (denoted by arrows) are shown under the boxes. Predicted binding motifs for other transcription factors are also indicated. The length of the bar is 100 bp. The WWCAAWG sequence is the consensus sequence for TBEs. (B) Alignment of proximal (TBE#1) and distal (TBE#2) TBE sequences. (C, D and E) Huh7 cells or HeLa cells were transfected with pGL3-<i>MENA</i> reporter plasmid and the promoter activity was stimulated by co-transfecting degradation-resistant β-catenin, Tcf4 or Wnt ligans or inhibited by co-transfecting dominant-negative Tcf4 (ΔNTcf4). The bars are given as averages (n = 3) normalized to Renilla luciferase activity, which was used an internal control. Error bars represent standard deviation (* = p<0.05, ** = p<0.01 and *** = p<0.005, Student's t-test). Each graph is a representative of at least two independent experiments. “β-cat” denotes the quadruple-mutant β-catenin-4m (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037013#s4" target="_blank">Materials & Methods</a>). (F) Chromatin immunoprecipitation assay was done with monoclonal anti-β-catenin antibody and PCR (35–40 cycles) was used to detect <i>MENA</i> promoter fragments. β-catenin was found to interact with <i>MENA</i> promoter in Huh7 and HEK293T cell context, as well as in mouse brain tissue and liver tissue contexts. All experiments were repeated at least twice. An unrelated monoclonal antibody was used as the control IgG. The lower bands that appear in the mouse brain gel image are the primer-dimer bands. The arrow indicates the band of the expected size.</p