Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

DNA-Binding and Photocleavage Properties of Ru(II) Polypyridyl Complexes with DNA and Their Toxicity Studies on Eukaryotic Microorganisms

Four Ru(II) polypyridyl complexes, [Ru(bpy)₂(7-NO₂-dppz)]²⁺, [Ru(bpy)₂(7-CH₃-dppz)]²⁺, [Ru(phen)₂(7-NO₂-dppz)]²⁺, and [Ru(phen)₂(7-CH₃-dppz)]²⁺ (bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline), (7-Nitro-dppz = 7-Nitro dipyrido[3,2-a:2′-3′-c]phenazine, 7-CH₃-dppz = 7-Methyl dipyrido[3,2-a:2′-3′-c]phenazine), have been synthesized and characterized by IR, UV, elemental analysis, ¹H NMR, ¹³C-NMR, and mass spectroscopy. The DNA-binding properties of the four complexes were investigated by spectroscopic and viscosity measurements. The results suggest that all four complexes bind to DNA via an intercalative mode. Under irradiation at 365 nm, all four complexes were found to promote the photocleavage of plasmid pBR 322 DNA. Toxicological effects of the selected complexes were performed on industrially important yeasts (eukaryotic microorganisms).     

LigandRNA: computational predictor of RNA–ligand interactions

RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA–small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA–ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a “meta-predictor” leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.     

Tumour necrosis factor-α and soluble Fas ligand as biomarkers in non-acetaminophen-induced acute liver failure

Arylamines and nitroarenes are very important intermediates in the industrial manufacture of dyes, pesticides and plastics, and are significant environmental pollutants. The metabolic steps of N-oxidation and nitroreduction to yield N-hydroxyarylamines are crucial for the toxic properties of arylamines and nitroarenes. Nitroarenes are reduced by microorganisms in the gut or by nitroreductases and aldehyde dehydrogenase in hepatocytes to nitrosoarenes and N-hydroxyarylamines. N-Hydroxyarylamines can be further metabolized to N-sulphonyloxyarylamines, N-acetoxyarylamines or N-hydroxyarylamine N-glucuronide. These highly reactive intermediates are responsible for the genotoxic and cytotoxic effects of this class of compounds. N-Hydroxyarylamines can form adducts with DNA, tissue proteins, and the blood proteins albumin and haemoglobin in a dose-dependent manner. DNA and protein adducts have been used to biomonitor humans exposed to such compounds. All these steps are dependent on enzymes, which are present in polymorphic forms. This article reviews the metabolism of arylamines and nitroarenes and the biomonitoring studies performed in animals and humans exposed to these substances.     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Lemon peel and Limoncello liqueur: A proteomic duet

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of lemon peels and pulp, of a home-made alcoholic infusion of peels and of a very popular Italian liqueur called “Limoncello”, stated to be an infusion of the flavedo (the outer, yellow skin of lemons). The aim of this study was not only to perform the deepest investigation so far of the lemon peel proteome but also to assess the genuineness of the commercial liqueur via a three-pronged attack. First, different extraction techniques have been used for the characterization of the peel (and additionally of the pulp) proteome, secondly a home-made infusion has been analysed and finally the proteome of the commercial drink was checked. The peel (the flavedo, not the underlying layer called albedo) proteome has been evaluated via prior capture with CPLLs at different pH values (2.2 and 7.2). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 1011 unique gene products in the peel extracts and 674 in the pulp, 264 proteins in the home-made infusion and just 8 proteins (and protein fragments), together with 12 peptides, in one Italian Limoncello produced in the Sorrento Region, thus proving the genuineness of this product. On the contrary, cheaper Limoncellos were devoid of any protein/peptide, casting doubts on their production from vegetable extracts. This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.     

The mechanism of high affinity pentasaccharide binding to antithrombin,insights from Gaussian accelerated molecular dynamics simulations

Abstract

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.

Communicated by Ramaswamy H. Sarma     

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

Novel insights into the pleiotropic effects of human serum albumin in health and disease

Background

Human serum albumin is the principal protein in human serum. It participates in regulation of plasma oncotic pressure and transports endogenous and exogenous ligands such as thyroxine, free fatty acids, bilirubin, and various drugs. Therefore, studying its ligand binding mechanism is important in understanding many functions of the protein.

Scope of review

This review discusses the pleiotropic biochemical effects and their relevance to physiologic functions of albumin.

Major conclusions

Although HSA is traditionally recognized for its ligand transport and oncotic effects in human circulation, our studies have revealed its participation in several other important physiological functions. In some instances, it may function as a catalyst. Pleiotropic properties of HSA have been exploited by development of recombinant HSA and its mutants, and the use of these recombinant proteins in studies with various biochemical and biophysical techniques. These studies allowed us to obtain new insights on the diverse roles of HSA in human physiology. The following aspects of HSA were discussed in this review: 1) HSA and its mutants' role in thyroxine transport, 2) structural details of the ligand binding functions of HSA to ligands such as warfarin, digoxin, halothane anesthetics, nitric oxide, bilirubin, free fatty acids, etc, and 3) the formation of modified albumin during myocardial ischemia, its diagnostic significance, and HSA's role in cardiovascular disease.

General significance

The appreciation and understanding of structural details and new physiological roles has provided a renewed interest in HSA research. Specific structural information gained on various mechanisms of HSA–ligand interaction can be used to develop a model to better understand protein–drug interactions, aid in the development of new drugs with improved pharmacokinetic effects, and ultimately be used to improve the quality of healthcare. This article is part of a Special Issue entitled Serum Albumin.