Extracting binding energies and binding modes from biomolecular simulations of fragment binding to endothiapepsin.

Fragment-based drug discovery (FBDD) aims to discover a set of small binding fragments that may be subsequently linked together. Therefore, in-depth knowledge of the individual fragments' structural and energetic binding properties is essential. In addition to experimental techniques, the direct simulation of fragment binding by molecular dynamics (MD) simulations became popular to characterize fragment binding. However, former studies showed that long simulation times and high computational demands per fragment are needed, which limits applicability in FBDD. Here, we performed short, unbiased MD simulations of direct fragment binding to endothiapepsin, a well-characterized model system of pepsin-like aspartic proteases. To evaluate the strengths and limitations of short MD simulations for the structural and energetic characterization of fragment binding, we predicted the fragments' absolute free energies and binding poses based on the direct simulations of fragment binding and compared the predictions to experimental data. The predicted absolute free energies are in fair agreement with the experiment. Combining the MD data with binding mode predictions from molecular docking approaches helped to correctly identify the most promising fragments for further chemical optimization. Importantly, all computations and predictions were done within 5 days, suggesting that MD simulations may become a viable tool in FBDD projects.

Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations.

Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Förster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.

Brain function and metabolism in patients with long-term tacrolimus therapy after kidney transplantation in comparison to patients after liver transplantation.

BACKGROUND: About 50% of the patients 5-7 years after kidney transplantation show impairment of memory, attention and executive function. Tacrolimus frequently induces neurological complications in the first few weeks after transplantation. Furthermore, tacrolimus treatment is associated with impaired cognitive function in the long-term in patients after liver transplantation. We hypothesize that long-term tacrolimus therapy is associated with cognitive dysfunction and alterations of brain structure and metabolism in patients after kidney transplantation. METHODS: Twenty-one patients 10 years after kidney transplantation underwent cognitive testing, magnetic resonance imaging and whole brain 31-phosphor magnetic resonance spectroscopy for the assessment of brain function, structure and energy metabolism. Using a cross-sectional study design the results were compared to those of patients 1 (n = 11) and 5 years (n = 10) after kidney transplantation, and healthy controls (n = 17). To further analyze the share of transplantation, tacrolimus therapy and kidney dysfunction on the results patients after liver transplantation (n = 9) were selected as a patient control group. RESULTS: Patients 1 and 10 years after kidney transplantation (p = 0.02) similar to patients 10 years after liver transplantation (p<0.01) showed significantly worse cognitive function than healthy controls. In contrast to patients after liver transplantation patients after kidney transplantation showed significantly reduced adenosine triphosphate levels in the brain compared to healthy controls (p≤0.01). Patients 1 and 5 years after kidney transplantation had significantly increased periventricular hyperintensities compared to healthy controls (p<0.05). CONCLUSIONS: Our data indicate that cognitive impairment in the long-term after liver and kidney transplantation cannot exclusively be explained by CNI neurotoxicity.

Altered neurometabolism in major depressive disorder: A whole brain 1H-magnetic resonance spectroscopic imaging study at 3T.

INTRODUCTION: Major depressive disorder (MDD) is a severe mental disorder with a neurobiological basis that is poorly understood. Several studies demonstrated widespread, functional and neurometabolic alterations in MDD. However, little is known about whole brain neurometabolic alterations in MDD. METHOD: Thirty-two patients with MDD and 32 paired on a one-to-one basis healthy controls (CTRL) underwent 1H-whole brain spectroscopic (1H-WBS) imaging. Lobar and cerebellar metabolite concentrations of brain N-acetylaspartate (NAA), total choline (tCho), total creatine (tCr), glutamine (Gln), glutamate (Glu), and myo-Inositol (mI) were assessed in patients and controls. RESULTS: Decreased NAA, tCho, and tCr were found in the right frontal and right parietal lobe in MDD compared to CTRL, and to a lesser extent in the left frontal lobe. Furthermore, in MDD increased glutamine was observed in the right frontal lobe and bitemporal lobes, and increased glutamate in the cerebellum. CONCLUSION: Altered global neurometabolism examined using 1H-WBS imaging in MDD may be interpreted as signs of neuronal dysfunction, altered energy metabolism, and oligodendrocyte dysfunction. In particular, the parallel decrease in NAA, tCr and tCho in the same brain regions may be indicative of neuronal dysfunction that may be counterbalanced by an increase of the neuroprotective metabolite glutamine. Future prospective investigations are warranted to study the functional importance of these findings.

Regional Metabolite Concentrations in Aging Human Brain: Comparison of Short-TE Whole Brain MR Spectroscopic Imaging and Single Voxel Spectroscopy at 3T.

PURPOSE: The aim of this study was to compare a recently established whole brain MR spectroscopic imaging (wbMRSI) technique using spin-echo planar spectroscopic imaging (EPSI) acquisition and the Metabolic Imaging and Data Analysis System (MIDAS) software package with single voxel spectroscopy (SVS) technique and LCModel analysis for determination of relative metabolite concentrations in aging human brain. METHODS: A total of 59 healthy subjects aged 20-70 years (n ≥ 5 per age decade for each gender) underwent a wbEPSI scan and 3 SVS scans of a 4 ml voxel volume located in the right basal ganglia, occipital grey matter and parietal white matter. Concentration ratios to total creatine (tCr) for N­acetylaspartate (NAA/tCr), total choline (tCho/tCr), glutamine (Gln/tCr), glutamate (Glu/tCr) and myoinositol (mI/tCr) were obtained both from EPSI and SVS acquisitions with either LCModel or MIDAS. In addition, an aqueous phantom containing known metabolite concentrations was also measured. RESULTS: Metabolite concentrations obtained with wbMRSI and SVS were comparable and consistent with those reported previously. Decreases of NAA/tCr and increases of line width with age were found with both techniques, while the results obtained from EPSI acquisition revealed generally narrower line widths and smaller Cramer-Rao lower bounds than those from SVS data. CONCLUSION: The wbMRSI could be used to estimate metabolites in vivo and in vitro with the same reliability as using SVS, with the main advantage being the ability to determine metabolite concentrations in multiple brain structure simultaneously in vivo. It is expected to be widely used in clinical diagnostics and neuroscience.

Quantitative magnetic resonance imaging indicates brain tissue alterations in patients after liver transplantation.

PURPOSE: To investigate cerebral microstructural alterations in patients treated with calcineurin inhibitors (CNI) after orthotopic liver transplantation (OLT) using quantitative magnetic resonance imaging (qMRI) and a cross-sectional study design. METHODS: Cerebral qMRI was performed in 85 patients in a median 10 years after OLT compared to 31 healthy controls. Patients were treated with different dosages of CNI or with a CNI-free immunosuppression (CNI-free: n = 19; CNI-low: n = 36; CNI-standard: n = 30). T2-, T2*- and T2'- relaxation times, as well as apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were measured in brain gray and white matter by using the regions of interest method. RESULTS: In comparison to controls, patients revealed significantly increased T2, T2*, T2', ADC and reduced FA, predominantly in the frontal white matter, indicating microstructural brain alterations represented by increased free water (increased T2), reduced neuronal metabolism (increased T2') and a lower degree of spatial organization of the nervous fibers (reduced FA). CNI-low and CNI-free patients showed more alterations than CNI-standard patients. Analysis of their history revealed impairment of kidney function while under standard CNI dose suggesting that these patients may be more vulnerable to toxic CNI side-effects. CONCLUSION: Our findings suggest that the individual sensitivity to toxic side effects should be considered when choosing an appropriate immunosuppressive regimen in patients after liver transplantation.

Brain metabolic alterations in patients with long-term calcineurin inhibitor therapy after liver transplantation.

BACKGROUND: Calcineurin inhibitor (CNI) neurotoxicity after liver transplantation might be due to impairment of the cerebral metabolism. AIMS: To investigate CNI-related alterations of brain metabolite distributions and associations between cognitive function and brain metabolism in patients with long-term CNI treatment after liver transplantation. METHODS: Eighty-two patients (19 CNI free, 34 CNI low-dose and 29 standard-dose CNI immunosuppression) 10 years after liver transplantation and 32 adjusted healthy controls underwent nonlocalised brain phosphorus magnetic resonance spectroscopy (MRS) and single voxel proton MRS in the parietal white matter to estimate brain metabolite contents. The MRS results were correlated with psychometric data assessing cognitive function. RESULTS: Phosphorus metabolite concentrations with the exception of phosphocreatine (PCr) were reduced in patients compared to controls. Particularly, patients with low-dose CNI therapy showed a significant decrease in adenosine triphosphate (0.209 ± 0.012 vs 0.222 ± 0.010; P < 0.001) and a significant increase in PCr (0.344 ± 0.026 vs 0.321 ± 0.017; P < 0.001) compared to controls. Myo-Inositol in the CNI free group (2.719 ± 0.549 institutional unit [iu]) was significantly lower compared to controls (3.181 ± 0.425 iu; P = 0.02), patients on low-dose (3.130 ± 0.513 iu; P < 0.05) and standard-dose CNI therapy (3.207 ± 0.632 iu; P < 0.02). Glutamate and glutamine levels correlated negatively with cognitive function (Repeatable Battery for the Assessment of Neuropsychological Status Total Scale: R = -0.362, P = 0.029). CONCLUSION: Long-term CNI therapy after liver transplantation might be associated with alterations of brain metabolites.

Effects of Aging on the Human Brain: A Proton and Phosphorus MR Spectroscopy Study at 3T.

BACKGROUND AND PURPOSE: To investigate accumulative aging effects on neurometabolism in human brain and to collect a reference dataset. METHODS: Fifty-four healthy volunteers aged evenly between 22 and 73 years were studied using whole-brain 1 H-MR spectroscopic imaging in combination with 31 P-MRS at 3T. Global metabolite concentrations of brain N-acetylaspartate (NAA), total choline (tCho), and total creatine (tCr), as well as phosphocreatine (PCr), adenosine-5'-triphosphate (ATP), phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Pi) were determined. Fractional volumes of brain gray matter (FVGM), white matter (FVWM), and total tissue (FVTB, GM+WM) were also estimated. RESULTS: With age, NAA, ATP, and PME, as well as FVTB and FVGM decreased and tCho and FVWM increased linearly. Positive correlations were found between FVGM and global concentrations of NAA, ATP, PME, and Pi. CONCLUSION: Age-related accumulative metabolic changes in aging human brain correlated with reduced neuronal metabolic activity and density, reflected by decreased NAA, reduced mitochondrial activity by decreased ATP, and reduced membrane synthesis by decreased PME. These changes are associated with age-related decrease of neuronal volume. Global NAA and ATP might be used as surrogate biomarker for monitoring aging in human brain.

Effects of a 72 hours fasting on brain metabolism in healthy women studied in vivo with magnetic resonance spectroscopic imaging.

Adaptive response of human brain to stress plays a key role in maintaining health. Knowledge about how stress affects neurometabolism may help to understand adaptive stress responses, and distinguish maladaptation in neuropsychiatric disorders. In this study, neurometabolic responses to fasting stress in healthy women were investigated. Fifteen healthy females were examined for mood and cognition and using whole-brain MR spectroscopic imaging before and immediately after a 72-h fasting. Results were compared to 15 age-matched healthy females who did not taken part in fasting (non-fasting). Maps of the distributions in the brain of N-acetylaspartate (NAA), total choline (tCho), total creatine (tCr), glutamine/glutamate (Glx), and myo-Inositol (mI) were derived. Metabolite concentrations of each brain lobe and cerebellum measured before fasting were compared to those of post-fasting and non-fasting by repeated-measures ANOVA. After fasting, mood scores significantly increased. Glx decreased in all nine brain regions, tCho in eight, NAA in four and tCr in one, with Glx having the greatest change and the frontal lobes being the most affected brain region. In conclusion, fasting directly influences neurometabolism, and the adaptive brain response to maintain energy homeostasis under food deprivation in healthy women is associated with metabolite-selective and region-dependent changes of metabolite contents.

Physiological neuronal decline in healthy aging human brain - An in vivo study with MRI and short echo-time whole-brain (1)H MR spectroscopic imaging.

Knowledge of physiological aging in healthy human brain is increasingly important for neuroscientific research and clinical diagnosis. To investigate neuronal decline in normal aging brain eighty-one healthy subjects aged between 20 and 70years were studied with MRI and whole-brain (1)H MR spectroscopic imaging. Concentrations of brain metabolites N-acetyl-aspartate (NAA), choline (Cho), total creatine (tCr), myo-inositol (mI), and glutamine+glutamate (Glx) in ratios to internal water, and the fractional volumes of brain tissue were estimated simultaneously in eight cerebral lobes and in cerebellum. Results demonstrated that an age-related decrease in gray matter volume was the largest contribution to changes in brain volume. Both lobar NAA and the fractional volume of gray matter (FVGM) decreased with age in all cerebral lobes, indicating that the decreased NAA was predominantly associated with decreased gray matter volume and neuronal density or metabolic activity. In cerebral white matter Cho, tCr, and mI increased with age in association with increased fractional volume, showing altered cellular membrane turn-over, energy metabolism, and glial activity in human aging white matter. In cerebellum tCr increased while brain tissue volume decreased with age, showing difference to cerebral aging. The observed age-related metabolic and microstructural variations suggest that physiological neuronal decline in aging human brain is associated with a reduction of gray matter volume and neuronal density, in combination with cellular aging in white matter indicated by microstructural alterations and altered energy metabolism in the cerebellum.

A membrane-proximal, C-terminal α-helix is required for plasma membrane localization and function of the G Protein-coupled receptor (GPCR) TGR5.

The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of ß-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the ß2 adrenergic receptor (ß2AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5ß2AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the ß2AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

α5 ß1-integrins are sensors for tauroursodeoxycholic acid in hepatocytes.

UNLABELLED: Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in the perfused rat liver in an α5 ß1 integrin-mediated way. However, the molecular basis of TUDC-sensing in the liver is unknown. We herein show that TUDC (20 µmol/L) induces in perfused rat liver and human HepG2 cells the rapid appearance of the active conformation of the ß1 subunit of α5 ß1 integrins, followed by an activating phosphorylation of extracellular signal-regulated kinases. TUDC-induced kinase activation was no longer observed after ß1 integrin knockdown in isolated rat hepatocytes or in the presence of an integrin-antagonistic hexapeptide in perfused rat liver. TUDC-induced ß1 integrin activation occurred predominantly inside the hepatocyte and required TUDC uptake by way of the Na(+) /taurocholate cotransporting peptide. Molecular dynamics simulations of a 3D model of α5 ß1 integrin with TUDC bound revealed significant conformational changes within the head region that have been linked to integrin activation before. CONCLUSIONS: TUDC can directly activate intrahepatocytic ß1 integrins, which trigger signal transduction pathways toward choleresis. (HEPATOLOGY 2013).

Molecular modelling studies of new potential human DNA polymerase α inhibitors.

The human polymerase α (pol α) is a promising target for the therapy of cancer e.g. of the skin. The authors recently built a homology model of the active site of human DNA pol α. This 3D model was now used for molecular modelling studies with eight novel analogues of 2-butylanilino-dATP, which is a highly selective nucleoside inhibitor of mammalian pol α. Our results suggest that a higher hydrophobicity of a carbohydrate side chain (pointing into a spacious hydrophobic cavity) may enhance the strength of the interaction with the target protein. Moreover, acyclic acyclovir-like derivatives outperformed those with a sugar-moiety, indicating that structural flexibility and higher conformational adaptability has a positive effect on the receptor affinity. Cytotoxicity tests confirmed our theoretical findings. Besides, one of our most promising compounds in the molecular modelling studies revealed high selectivity for the SCC-25 cell line derived from squamous cell carcinoma in man.