GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

<p>Abstract</p> <p>Background</p> <p>GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective <it>in vivo </it>knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor.</p> <p>Results</p> <p>Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found.</p> <p>Conclusion</p> <p>Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.</p

Axial Imidazole Distorsion Effects on the Catalytic and Binding Properties of Chelated Deuterohemin Complexes

The effect of strain in the axial coordination of imidazole to the heme has been studied in the chelate complexes deuterohemin-histidine (DH-His) and deuterohemin-alanylhistidine (DH-AlaHis). Molecular mechanics calculations indicate that three types of distortion of the axial ligand occur in DH-His, due to the relatively short length of the arm carrying the donor group: tilting off-axis, tipping, and inclination of the imidazole plane with respect to the axial Fe-N bond. The effects of tilting (¢ç 10°) and inclination of the imidazole ring (¢ä 17°) are dominant, while tipping is small and is probably of little importance here. By contrast, the axial imidazole coordination is normal in DH-AlaHis and other computed deuterohemin-dipeptide or -tripeptide complexes where histidine is the terminal residue, the only exception being DH-ProHis, where the rigidity of the proline ring reduces the flexibility of the chelating arm. The distortion in the axial iron-imidazole bond in DH-His has profound and negative influence on the binding and catalytic properties of this complex compared to DH-AlaHis. The former complex binds more weakly carbon monoxide, in its reduced form, and imidazole, in its oxidized form, than the latter. The catalytic efficiency in peroxidative oxidations is also reduced in DH-His with respect to DH-AlaHis. The activity of the latter complex is similar to that of microperoxidase-11, the peptide fragment incorporating the heme that results from hydrolytic cleavage of cytochrome

The Dual Behaviour of a GPCR Involved in Brain Damage an Repair: Forced Unbinding of the Receptor GPR17 Ligands from Wild Type and R255I Mutant Models Through a Computational Approach

GPR17 is a hybrid G-protein-coupled receptor activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes, and involved in brain damage and repair. Its exploitment as a target for novel neuroreparative strategies depends on the elucidation of the molecular determinants driving binding of its ligands. We applied docking and molecular dynamics simulations to analyse the binding and the forced unbinding of two GPR17 ligands (the purinergic agonist UDP and the leukotriene receptor antagonist pranlukast) from both the wild-type receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I). Molecular dynamics suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and EL2. The driving interaction involves R255 and the UDP phosphate moiety. Steered molecular dynamics experiments showed that the energy required to unbind UDP is higher for the wild-type receptor than for R255I. Three potential binding sites for pranlukast were found. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the wild-type receptor and the R255I receptor were found for the unbinding of pranlukast. These data suggest a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interst for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase\u2013ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible

Annex 65, Long-Term Performance of Super-Insulating-Materials in Building Components and Systems. Report of Subtask III: Practical Applications – Retrofitting at the Building Scale – Field scale

More than 80% of the energy consumption will be influenced by the existing building stock. Accordingly, building renovation has a high priority in many countries. Furthermore, several studies have shown that the most efficient way to curb the energy consumption in the building sector (new &amp; existing) remain the reduction of the heat loss by improving the insulation of the building envelope (roof, floor, wall &amp; windows). All since the first oil crisis in 1973-1974, the national building regulations require improvement of the thermal performance of the building envelope to significantly reduce the energy use for space heating. Following the regulations, the energy efficiency of new buildings has improved. In Europe, targeting to an average U-value close to 0.2 W/m2\ub7K is optimal. Using traditional insulation materials this means an insulation thickness of about 20 cm. Thus, the thickness of internal and/or external insulation layers becomes a major issue of concern for retrofitting projects and even for new building projects in cities. Therefore, there is a growing interest in the so-called super-insulating materials (SIM). The scope of the present work covers two different types of SIMs:• Advanced Porous Materials (APM), where the gaseous heat transfer is hindered significantly by the fine structure in the sub-micrometre range, and• Vacuum Insulation Panels (VIP), where the contribution of gaseous conductivity to the total heat transfer is suppressed by evacuation.For Advanced Porous Materials (APM) one might distinguish between• porous silica e.g. based on fumed silica, and• aerogels.For Vacuum Insulation Panels (VIP) one might distinguish between:• different core materials: fumed silica, glass fibre, PU, EPS, others;• different envelopes: metalized film, aluminium laminate, stainless steel, glass, or combinations;• with or without a getter and/or a desiccant.The objective of this Annex 65 Subtask 3 report is to define the application areas of SIM and to describe the conditions of the intended use of the products. Indeed, it’s clear that the requested performance of the SIM will strongly depend on the temperature, humidity and load conditions. For building applications, storage, handling and implementation requirements are also described. Common and specific numerical calculations will be performed at the building scale to assess the impact of SIM on the performance of the building envelope.SIM was used in almost all building components with different environmental condition (boundary condition) and in different climate zone. The moisture and temperature conditions in building components can cause moisture/temperature induced stresses and the stresses can cause damage in sensitive super insulation material e.g. VIPs. Thus, to convince the conservative market of construction, it needs, in addition to laboratory measurements, real condition/environmental measurements of commercially realized objects (new buildings as well as refurbishments) under several years of operation.The long-term performance of super insulation materials has to be determined based on case studies in field and laboratory. Full scale experiments provide knowledge of practical and technical difficulties as well as data for service life estimation. For certain conclusions to be drawn from the case studies, monitoring is essential. Unfortunately, monitoring is only performed in few case studies. In this report these experiences are gathered and evaluated from a long-term performance perspective.APMs have been commercially successful in the building industry in niche applications typically with space restrictions since the early 2000s. Therefore, over the last years, a number of state-of-the-art reviews have focused on applications of advanced porous materials, such as aerogels, used as thermal insulation in buildings. VIPs, on the other hand, have also been used in other applications than buildings, such as refrigerators and transport boxes. The different applications areas have been identified by numerous researchers. However, in most studies of VIPs available in the literature, it was only the thermal performance of the assembly that was investigated. However, also the moisture performance is important to consider since changes to existing structures will influence the risk for moisture damages.In the Annex, the gathered case studies cover a wider range of SIM i.e. aerogel blankets, AB, (7 case studies), silica-based boards, SB, (3 case studies) and VIP (22 case studies). The aim was to gather information from projects where SIMs were used in different assemblies. Some of the projects have been monitored, i.e. sensors were installed to monitor the temperature, relative humidity or heat flux through the assemblies, while only three have been followed up, i.e. where a third party have analysed the results of the monitoring. The case studies are presented and specific and general conclusions from each application are made.The case studies showed that aerogel blankets are possible to install in up to five layers (50 mm) without too much difficulty. The evaluations showed that the performance of the aerogel blankets was maintained over the evaluation period. For VIPs, it is difficult to evaluate the performance when installed in the wall. In one of the case studies in the report, the external air space made it impossible to identify the different panels by thermography. Only indirect methods, like evaluation of the measured temperatures in the wall, can be used to follow the long-term performance of the panels. In another case study, hybrid insulated district heating pipes were installed at two locations in a district heating system with temperatures up to 90\ub0C. Measurements during the period 2012 to 2015 showed no sign of deterioration of the VIPs and the temperature profile over the pipes was constant. An existing masonry wall was insulated with VIP-foam sandwich (XPS-VIP-XPS). It showed satisfactory and promising performance for a period of six years (2011-present). The analysis of the data obtained from continuous temperature monitoring across each insulation layer indicated the aging of VIP remains insignificant.In the framework of IEA EBC Annex 65 a common simulation-based procedure was introduced with the scope to identify potential critical hygrothermal working conditions of the SIM, which were identified as main drivers of the ageing effect. The study highlights that some physical phenomena (such as thermal bridging effects, the influence of temperature on the thermal conductivity and the decay of performance over time depending on the severity of the boundaryconditions) should be carefully evaluated during the design phase in order to prevent the mismatch between expected/predicted and the actual thermal performance.As general guidelines to mitigate the severity of the operating conditions of VIP, a list of recommendation are in the following summarised:• For the external wall insulation with VIP in solar exposed fa\ue7ade, the adoption of ventilated air layer could dramatically reduce the severity of the VIP operating conditions. Alternatively, light finishing colour are warmly encouraged to mitigate the surface temperature.• The protection of VIP with thin traditional insulation layer is always encouraged.• The application of VIP behind heater determines high value of surface temperature field which could potentially lead to a fast degradation of the panel. A possible solution to mitigate the severity of the boundary conditions could be the coupling of VIP with a radiant barrier, or the protection of VIP with thin insulation layer when it is possible.• In roof application, light colour (cool roof), performant water proof membrane, ventilated airspace and gravel covering layer (flat roof) represent effective solutions to mitigate the severe exposure.• In presence of wall subjected to high driving rain, it is preferable to adopt ventilated fa\ue7ade working as rain-screen to prevent the water absorption.Furthermore, to provide designers, engineers, contractors and builders with guidelines for the applications of vacuum insulation panels (VIPs) and Advanced Porous Materials (APMs) examples are given of methods that may be used to verify the quality and thermal performance of SIMs after installation. A comprehensive account of transport, handling, installation and quality check precures are presented. The main purpose of the descriptions is to promote safe transport, handling and installation. In the case of VIPs the primary issue is that of protecting the panels whereas the main concern for APMs is the safety in handling of the material.During the work of the Annex several questions regarding the long-term performance of SIMs on the building scale have been identified and discussed. Four main challenges were identified:• Knowledge and awareness among designers concerning using SIM• Conservative construction market• Cost versus performance• Long-term performance of SIMsFinally, SIMs for building applications have been developed in the recent decades. Theoretical considerations and first practical tests showed that VIP, especially those with fumed silica core, are expected to fulfil the requirements on durability in building applications for more than 25 years. Both VIPs and APMs have been successfully installed over the past 15 years in buildings. However, real experience from practical applications exceeding 15 years is still lacking, especially when considering third-party monitoring and follow up of demonstrations

Electronic and geometrical structures of nitroso ligands coordinated to transition-metal atoms. A nonempirical theoretical study of bis(phosphino)(nitrosotrifluoromethane)platinum and bis(phosphine)(nitrosomethane)platinum

The modifications of the molecular geometry and electronic structure of CF3NO and CH3NO caused by coordination to a Pt(0) center have been studied by means of nonempirical MO-LCAO theoretical calculations on the model complexes Pt(PH3)2(RNO). The \u3b72 coordination mode of RNO, in which the N-O group acts as a side-on \u3c3-donor and \u3c0-acceptor, is found to be more favorable than the \u3b71 mode, in which only the N atom directly interacts with the metal center. The bonding characteristics of the RNO ligands are compared with those of the isoelectronic dioxygen ligand by investigating the electronic structure of the Pt(PH3)2(\u3b72O2) complex

Tailoring Transition Metal Complexes for Nonlinear Optics Applications. 2. A Theoretical Investigation of the Second-Order Nonlinear Optical Properties of M(CO)5L Complexes (M = Cr, W; L = Py, PyCHO, Pyz, PyzBF3, BPE, BPEBF3)

The authors report an ab initio study of 2nd-order nonlinear optical (NLO) properties and absorption electronic spectra of push-pull transition metal chromophores [M(CO)5L] (M = Cr, W; L = pyridine (Py), 4-formyl-pyridine (PyCHO), pyrazine (Pyz), trans-1,2-bis(4-pyridyl)ethylene (BPE)). Pyz and BPE are considered either with 1 N atom free or interacting with the strong acceptor BF3. All of the mol. properties were calcd. using 2 different and methodol. independent approaches: the time dependent and coupled perturbed d. functional theories (TDDFT and CPDFT) and the sum-over-states (SOS) approach, where the excited states are obtained via the single CI (SCI) ab initio method. DFT results are in acceptable agreement with the exptl. energy values of electronic transitions (with the exception of chromophores with the large greek p-delocalization, like BPE); SCI calcns. overestimate excitation energies and produce an inversion in the order of dM greek pi*L and dM greek p*CO transitions. The SCI-SOS approach gives 1st-order hyperpolarizabilities, basically in agreement as trend and values with the expts. and seems to be a tool generally suitable for the evaluation of these properties also for transition metal complexes. However, the 1st-order hyperpolarizabilities computed using the CPDFT approach are consistently overestimated in comparison with the exptl. results, esp. in the case of a ligand with large greek p-delocalization. Also the 2-level approxn. taking into account only the lowest energy charge transfer excitation (e.g., dM greek p*L) is not applicable to chromophores with the extended greek p-delocalized ligand (BPE) coordinated to a transition metal, due to significant contributions originating from intraligand greek pL greek p*L transitions. This study reports a detailed anal. and comparison of electronic NLO effects of transition metal complexes computed with DFT and ab initio SCI-SOS methodol