Ribavirin inhibits the replication of infectious bursal disease virus predominantly through depletion of cellular guanosine pool

IntroductionThe antiviral activity of different mutagens against single-stranded RNA viruses is well documented; however, their activity on the replication of double-stranded RNA viruses remains unexplored. This study aims to investigate the effect of different antivirals on the replication of a chicken embryo fibroblast-adapted Infectious Bursal Disease virus, FVSKG2. This study further explores the antiviral mechanism utilized by the most effective anti-IBDV agent.MethodsThe cytotoxicity and anti-FVSKG2 activity of different antiviral agents (ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride) were evaluated. The virus was serially passaged in chicken embryo fibroblasts 11 times at sub-cytotoxic concentrations of ribavirin, 5-fluorouracil or amiloride. Further, the possible mutagenic and non-mutagenic mechanisms utilized by the most effective anti-FVSKG2 agent were explored.Results and DiscussionRibavirin was the least cytotoxic on chicken embryo fibroblasts, followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin inhibited the replication of FVSKG2 in chicken embryo fibroblasts significantly at concentrations as low as 0.05 mM. The extinction of FVSKG2 was achieved during serial passage of the virus in chicken embryo fibroblasts at ≥0.05 mM ribavirin; however, the emergence of a mutagen-resistant virus was not observed until the eleventh passage. Further, no mutation was observed in 1,898 nucleotides of the FVSKG2 following its five passages in chicken embryo fibroblasts in the presence of 0.025 mM ribavirin. Ribavarin inhibited the FVSKG2 replication in chicken embryo fibroblasts primarily through IMPDH-mediated depletion of the Guanosine Triphosphate pool of cells. However, other mechanisms like ribavirin-mediated cytokine induction or possible inhibition of viral RNA-dependent RNA polymerase through its interaction with the enzyme’s active sites enhance the anti-IBDV effect. Ribavirin inhibits ds- RNA viruses, likely through IMPDH inhibition and not mutagenesis. The inhibitory effect may, however, be augmented by other non-mutagenic mechanisms, like induction of antiviral cytokines in chicken embryo fibroblasts or interaction of ribavirin with the active sites of RNA-dependent RNA polymerase of the virus

KAISO inhibition: an atomic insight

<div><p>In today’s world, the pursuit of a novel anti-cancer agent remains top priority because of the fact that the global burden of this malady is continuously increasing. Our work is no different from others in searching for new therapeutic solutions. To achieve this, we are looking into Epigenetics, the phenomenon governed by hypermethylation and hypomethylation of tumor suppressor genes and oncogenes, respectively. Our target for this study is an important intermediary methyl-CpG binding protein named kaiso. In our study, we have used the X-ray crystallographic structure of Kaiso for virtual screening and molecular dynamics simulations to study the binding modes of possible inhibitors. The C2H2 domain comprising LYS539 was used for screening the inter bio screen Database having 48,531 natural compounds. Our approach of using computer-aided drug designing methods helped us to remove the execrable compounds and narrowed our focus on a selected few for molecular simulation studies. The top ranked compound (chem. ID 28127) exhibited the highest binding affinity and was also found to be stable throughout the 20 ns timeframe. This compound is therefore a good starting point for developing strong inhibitors.</p></div

A novel kinase mutation in VEGFR-1 predisposes its alpha C-helix/activation loop towards allosteric activation: Atomic insights from protein simulation

Vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in diverse pathologies, including cancers. Although VEGFR-1 is considered as functionally impaired kinase, its decoy characteristics make it an important regulator of VEGFR-mediated signaling, particularly in tumor angiogenesis. VEGFR-1 conveys signaling via its tyrosine kinase (TK) domain whose activation is regulated by phosphorylation of specific tyrosine residues. Thus dysregulation of VEGFR-1 signaling, as reported in most of the cancers, might be a consequence of altered phosphorylation that could be attributed to genotypic variations in its TK domain. Considering the importance of TK domain of VEGFR-1, we carried out its mutational screening in 84 clinically validated and histopathologically confirmed colorectal cancer patients. By means of direct DNA sequencing and SNP analyses, eight novel variations, including one synonymous, two deletion, one missense and four intronic variations, were reported in the TK domain of VEGFR-1. rs730882263:C>G variation specifically reported in colon cancer, representing a single-atomic change (Sulfur to Oxygen) in the predicted (p.Cys1110Ser) protein, was observed as potentially deleterious variation as assessed by multiple single-nucleotide polymorphism prediction servers. Molecular dynamics simulations of VEGFR-1 Wt and (p.Cys1110Ser) variant models revealed major conformational changes in variant protein presumptuously generating an open conformation thereby exposing the activation domain and consequently increasing the probability of phosphorylation events: a condition frequently reported in cancers

Atomic Insight into the Altered O⁶-Methylguanine-DNA Methyltransferase Protein Architecture in Gastric Cancer

<div><p>O⁶-methylguanine-DNA methyltransferase (MGMT) is one of the major DNA repair protein that counteracts the alkalyting agent-induced DNA damage by replacing O⁶-methylguanine (mutagenic lesion) back to guanine, eventually suppressing the mismatch errors and double strand crosslinks. Exonic alterations in the form of nucleotide polymorphism may result in altered protein structure that in turn can lead to the loss of function. In the present study, we focused on the population feared for high exposure to alkylating agents owing to their typical and specialized dietary habits. To this end, gastric cancer patients pooled out from the population were selected for the mutational screening of a specific error prone region of MGMT gene. We found that nearly 40% of the studied neoplastic samples harbored missense mutation at codon¹⁵¹ resulting into Serine to Isoleucine variation. This variation resulted in bringing about the structural disorder, subsequently ensuing into a major stoichiometric variance in recognition domain, substrate binding and selectivity loop of the active site of the MGMT protein, as observed under virtual microscope of molecular dynamics simulation (MDS). The atomic insight into MGMT protein by computational approach showed a significant change in the intra molecular hydrogen bond pattern, thus leading to the observed structural anomalies. To further examine the mutational implications on regulatory plugs of MGMT that holds the protein in a DNA-Binding position, a MDS based analysis was carried out on, all known physically interacting amino acids essentially clustered into groups based on their position and function. The results generated by physical-functional clustering of protein indicated that the identified mutation in the vicinity of the active site of MGMT protein causes the local and global destabilization of a protein by either eliminating the stabilizing salt bridges in cluster C3, C4, and C5 or by locally destabilizing the “protein stabilizing hing” mapped on C3-C4 cluster, preceding the active site.</p></div

Illustrative representation of drastic conformational variability that a mutant structure (arrow specifies the site of an mutated residue) undergoes when compared wild type protein structure.

<p>The snapshots were retrieved at every 5 ns interval along the 30 ns simulation.</p

Intra protein hydrogen bond profile of wt and Mu MGMT protein over time at 300K.

<p>Intra protein hydrogen bond profile of wt and Mu MGMT protein over time at 300K.</p

(a) Two dimensional representation of the motion of both structures along the first two principal eigenvectors, Black and green represent the wt and Mu MGMT respectively.

<p>(b) FEL of both the motions generated separately. (c) Separate two dimensional representations of PCA of both wt and Mu MGMT with the inset of three most stable structures at different point of time.</p

Three dimensional time dependent Phi/Psi distribution showing the change in the Gibbs free energy by the mutation in the Clusters 3 and 4 (black = wt, Red = Mu).

<p>Three dimensional time dependent Phi/Psi distribution showing the change in the Gibbs free energy by the mutation in the Clusters 3 and 4 (black = wt, Red = Mu).</p

Representative picture of modeled wild type MGMT protein docked to minor groove of DNA depicts a stable interaction between Ser151 of enzyme with thymine base, attaining stability with the help of two hydrogen bonds shown in the figure as dotted arrows.

<p>Representative picture of modeled wild type MGMT protein docked to minor groove of DNA depicts a stable interaction between Ser151 of enzyme with thymine base, attaining stability with the help of two hydrogen bonds shown in the figure as dotted arrows.</p

(a) Protein RMSDs for wt and Mu MGMT structures at 300 K. wt is shown in black and Mu in green.

<p>(Insets A and B) shows the relative structures at the point of RMSD jump. (b) MGMT residue RMSF along the MDS and the arrow pointing out to the region showing maximum fluctuation. (c) The RMSD vs. Atomic units. Plot showing highly unstable Mu curve in red.</p