Evaluation of a novel virtual screening strategy using receptor decoy binding sites

Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated

Influenza A nucleoprotein binding sites for antivirals: current research and future potential

This document is the Accepted Manuscript version of the following article: Andreas Kukol and Hershna Patel, ‘Influenza A nucleoprotein binding sites for antivirals: current research and future potential’, Future Biology, Vol 9(7): 625-627, July 2014. The version of record is available online at doi: 10.2217/fvl.14.45Peer reviewedFinal Accepted Versio

Evolutionary conservation of influenza A PB2 sequences reveals potential target sites for small molecule inhibitors.

The influenza A basic polymerase protein 2 (PB2) functions as part of a heterotrimer to replicate the viral RNA genome. To investigate novel PB2 antiviral target sites, this work identified evolutionary conserved regions across the PB2 protein sequence amongst all sub-types and hosts, as well as ligand binding hot spots which overlap with highly conserved areas. Fifteen binding sites were predicted in different PB2 domains; some of which reside in areas of unknown function. Virtual screening of ~50,000 drug-like compounds showed binding affinities of up to 10.3 kcal/mol. The highest affinity molecules were found to interact with conserved residues including Gln138, Gly222, Ile529, Asn540 and Thr530. A library containing 1738 FDA approved drugs were screened additionally and revealed Paliperidone as a top hit with a binding affinity of -10 kcal/mol. Predicted ligands are ideal leads for new antivirals as they were targeted to evolutionary conserved binding sites

Large-scale analysis of influenza A virus nucleoprotein sequence conservation reveals potential drug-target sites

The nucleoprotein (NP) of the influenza A virus encapsidates the viral RNA and participates in the infectious life cycle of the virus. The aims of this study were to find the degree of conservation of NP among all virus subtypes and hosts and to identify conserved binding sites, which may be utilised as potential drug target sites. The analysis of conservation based on 4430 amino acid sequences identified high conservation in known functional regions as well as novel highly conserved sites. Highly variable clusters identified on the surface of NP may be associated with adaptation to different hosts and avoidance of the host immune defence. Ligand binding potential overlapping with high conservation was found in the tail-loop binding site and near the putative RNA binding region. The results provide the basis for developing antivirals that may be universally effective and have a reduced potential to induce resistance through mutations.Peer reviewe

G-quadruplex formation of FXYD1 pre-mRNA indicates the possiblity of regulating expression of its protein product

G-quadruplexes are higher-order nucleic acid structures formed of square-planar arrangements of four guanine bases held together by Hoogsteen-type hydrogen bonds. Stacks of guanine tetrads are stabilised by intercalating potassium ions. FXYD1 encodes for phospholemman, a regulatory subunit of the cardiac Na+/K+-ATPase. Computational sequence analysis of FXYD1 pre-mRNA predicted the formation of stable intramolecular G-quadruplexes in human and orthologue sequences. Multiple sequence alignment indicated that G-rich sequences are conserved in evolution suggesting a potential role of G-quadruplexes in FXYD1 gene expression. The existence of a non-functional alternative splicing product indicated that the G-quadruplex formation may control alternative splicing. Quadruplex formation of human and bovine oligonucleotides was confirmed in vitro by native polyacrylamide gel electrophoresis and intrinsic fluorescence emission spectroscopy. Taking together the evolutionary conservation of G-quadruplex forming sequences with the confirmation of G-quadruplex formation in vitro by two FXYD1 homologues the results point to a potential role of these structures in regulating the expression of FXYD1 and thus regulate indirectly the activity of the cardiac Na+/K+ -ATPase.Peer reviewe

A cellulose-based bioassay for the colorimetric detection of pathogen DNA

Cellulose-paper-based colorimetric bioassays may be used at the point of sampling without sophisticated equipment. This study reports the development of a colorimetric bioassay based on cellulose that can detect pathogen DNA. The detection was based on covalently attached single-stranded DNA probes and visual analysis. A cellulose surface functionalized with tosyl groups was prepared by the N,N-dimethylacetamide-lithium chloride method. Tosylation of cellulose was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and elemental analysis. Sulfhydryl-modified oligonucleotide probes complementary to a segment of the DNA sequence IS6110 of Mycobacterium tuberculosis were covalently immobilized on the tosylated cellulose. On hybridization of biotin-labelled DNA oligonucleotides with these probes, a colorimetric signal was obtained with streptavidin-conjugated horseradish peroxidase catalysing the oxidation of tetramethylbenzamidine by H2O2. The colour intensity was significantly reduced when the bioassay was subjected to DNA oligonucleotide of randomized base composition. Initial experiments have shown a sensitivity of 0.1 μM. A high probe immobilization efficiency (more than 90 %) was observed with a detection limit of 0.1 μM, corresponding to an absolute amount of 10 pmol. The detection of M. tuberculosis DNA was demonstrated using this technique coupled with PCR for biotinylation of the DNA. This work shows the potential use of tosylated cellulose as the basis for point-of-sampling bioassays.Peer reviewedFinal Accepted Versio

Structure and dynamics of the kinase IKK-β – A key regulator of the NF-kappa B transcription factor

'This is the author's version of a work that was accepted for publication in Journal of Structural Biology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Structural Biology, 176 (2) (2011) DOI 10.1016/j.jsb.2011.07.012'The inhibitor κB kinase-β (IKK-β) phosphorylates the NF-κB inhibitor protein IκB leading to the translocation of the transcription factor NF-κB to the nucleus. The transcription factor NF-κB and consequently IKK-β are central to signal transduction pathways of mammalian cells. The purpose of this research was to develop a 3D structural model of the IKK-β kinase domain with its ATP cofactor and investigate its dynamics and ligand binding potential. Through a combination of comparative modelling and simulated heating/annealing molecular dynamics (SAMD) simulation in explicit water the model accuracy could be substantially improved compared to comparative modelling on its own as shown by model validation measures. The structure revealed the details of ATP/Mg2+ binding indicating hydrophobic interactions with the adenine base and a significant contribution of Mg2+ as a bridge between ATP phosphate groups and negatively charged side chains. The molecular dynamics trajectories of the ATP-bound and free enzyme showed two conformations in each case, which contributed to the majority of the trajectory. The ATP-free enzyme revealed a novel binding site distant from the ATP binding site that was not encountered in the ATP bound enzyme. Based on the overall structural flexibility, it is suggested that a truncated version of the kinase domain from Ala14 to Leu265 should be subjected to crystallisation trials. The 3D structure of this enzyme will enable rational design of new ligands and analysis of protein–protein interactions. Furthermore, our results may provide a new impetus for wet-lab based structural investigation focussing on a truncated kinase domain.Peer reviewedFinal Accepted Versio

Composition of pigment complex in leaves of soybean plants, inoculated by Bradyrhizobium japonicum, subject to metal nanocarboxylates and various-levels of water supply

A distinctive feature of legumes is the ability to combine two most important processes: photosynthesis and nitrogen fixation. However, the course of those processes, and therefore seed potential of those crops depend on a number of biotic and abiotic factors, the commonest being drought. Therefore, interest in physical-biochemical resistance of the plant organism to abiotic stress factors is increasing, as well as search for optimum ways to increase its adaptability. Success of adaptation of a plant’s organism to unfavourable environmental factors is known to largely depend on optimal functioning of assimilative apparatus. Some indicators of the condition of the apparatus are the content and ratio of photosynthesis pigments. Therefore, we aimed at determining the reaction of the pigment complex of Glycine max (L.) Merr. plants, grown against the background of optimal and insufficient watering, to inoculation of seeds with rhizobia bacteria Bradyrhizobium japonicum, cultivated using nanocarboxylates of chromium, cobalt, iron, copper and germanium. Research has shown that utilization of germanium nanocarboxylate as a component of inoculative suspension led to the highest content of chlorophylls in leaves of soybean of the studied variants in the blossoming phase during optimal watering, as well as significant increase in the content of carotenoids compared with the control plants regardless of the level of watering. At the same time, this element caused no significant effect on the chlorophyll content in plants grown in drought. It was confirmed that among soybean plants that were in stress conditions (blossoming phase) for two weeks, the highest content of chlorophylls was in leaves of plants grown from seeds inoculated with rhizobial suspension with addition of chromium and copper nanocarboxylates, which caused 25.3% and 22.8% increase in chlorophyll а, 29.4% and 32.3% in chlorophyll b and 26.4%% and 23.8% in them respectively, compared with the control. Furthermore, chromium and copper nanocarboxylates stimulated the content of carotenoids in the same plants, though it was less expressed than after adding germanium nanocarboxylate. The highest content of photosynthetic pigments in plants after the watering was resumed (phase of bean formation) was in cases of applying chromium and germanium nanocarboxylates. It was confirmed that the most efficient way to protect the pigment complex of soybean plants during drought was using chromium and germanium nanocarboxylates as components of inoculation suspension. The results we obtained indicate the possibility of applying chromium nanocarboxylate in the technology of cultivating soybean in the conditions of water deficiency as an effective way to improve biosynthesis of chlorophylls, as well as using germanium nanocarboxyllate as a component that provides a high level of activity of protective mechanisms of the pigment system of soybean, associated with resisting stress caused by water deficiency