SYNTHESIS, INSILICO DOCKING AND ADMET STUDIES OF ARYL ACETIC ACID DERIVATIVES AS PROSTAGLANDIN ENDOPEROXIDE H SYNTHASE-2 INHIBITORS

Objectives: To study the inhibition of prostaglandin endoperoxide H synthase-2 (PHSH-2) for arylacetic acid derivatives. Methods: This study was performed to evaluate the anti-inflammatory activity of the synthesized arylacetic acid erivatives through molecular docking via Discovery Studio 4.0 and Schrodinger Software. ADMET study was conducted to find the assessment on genotoxicology.Results: The synthesized arylacetic acid derivatives were confirmed by nuclear magnetic resonance, liquid chromatography-mass spectrometry, and purity by high-performance liquid chromatography. The synthetic pathway is economical, industrial scalability and is achieved with high yield and purity. The in silico studies identified the active pocket and compared with the standard drug.Conclusion: Results from this work conclude that the arylacetic acid derivatives have very good inhibition and very low binding energy toward the active pocket, hence can be considered as good inhibitors of PHSH-2 on comparison with iodosuprofen. The compounds qualified Lipinski rule of five and the ADMET results were non-mutagenic and non-carcinogenic.Keywords: Arylacetic acid, 1 phenyl glycidyl ether protein, ADMET, In silico docking, Anti-inflammatory

Binding Modes of Peptidomimetics Designed to Inhibit STAT3

STAT3 is a transcription factor that has been found to be constitutively activated in a number of human cancers. Dimerization of STAT3 via its SH2 domain and the subsequent translocation of the dimer to the nucleus leads to transcription of anti-apoptotic genes. Prevention of the dimerization is thus an attractive strategy for inhibiting the activity of STAT3. Phosphotyrosine-based peptidomimetic inhibitors, which mimic pTyr-Xaa-Yaa-Gln motif and have strong to weak binding affinities, have been previously investigated. It is well-known that structures of protein-inhibitor complexes are important for understanding the binding interactions and designing stronger inhibitors. Experimental structures of inhibitors bound to the SH2 domain of STAT3 are, however, unavailable. In this paper we describe a computational study that combined molecular docking and molecular dynamics to model structures of 12 peptidomimetic inhibitors bound to the SH2 domain of STAT3. A detailed analysis of the modeled structures was performed to evaluate the characteristics of the binding interactions. We also estimated the binding affinities of the inhibitors by combining MMPB/GBSA-based energies and entropic cost of binding. The estimated affinities correlate strongly with the experimentally obtained affinities. Modeling results show binding modes that are consistent with limited previous modeling studies on binding interactions involving the SH2 domain and phosphotyrosine(pTyr)-based inhibitors. We also discovered a stable novel binding mode that involves deformation of two loops of the SH2 domain that subsequently bury the C-terminal end of one of the stronger inhibitors. The novel binding mode could prove useful for developing more potent inhibitors aimed at preventing dimerization of cancer target protein STAT3

Beiträge zur Glykobioinformatik Entwicklung von Software-Werkzeugen für die Glykobiologie

Die vorliegende Arbeit umfasst die Entwicklung von Algorithmen und Strategien zur Analyse von Massenspektren von Glykanen und Kohlenhydrate sowie Strategien zur voll- und halbautomatischen Aktualisierung und Annotierung einer bestehenden Datenbank der Sweet-DB. Für die Glykomik fehlte es bisher an Algorithmen, die ähnlich wie im Bereich der Proteomik bei der Sequenzierung von Peptiden, dem Benutzer eine Hilfe bei der Analyse von N-, O-Glykanen und Lipopolysacchariden sind. Die Zusammensetzung dieser Verbindungen ist aber für das Verständnis der zellulären Stoffwechsel-physiologie von essentieller Bedeutung. Im Rahmen der Entwicklung von Algorithmen zur Aufklärung von Massenspektren wurden insgesamt drei Programme entwickelt, die es dem Forscher gestatten, eine große Anzahl von Spektren, die im Bereich der Proteomik und Glykomik anfallen, auszuwerten. Dabei entstanden die Programme findYSeries, Glyco-Fragment und peakAssign, die eine schnellere Auswertung von Massenspektren im Bereich der Glykobiologie gestatten. So kann mit diesen Programmen die glykosylierte Aminosäure, die Komposition, die Anzahl der Antennen eines Glykans oder sogar die Sequenz eines Kohlenhydrats ermittelt werden. Im selben Maße wie der Bedarf an Programmen zur Auswertung von Messdaten zunimmt, steigt auch die Menge der daraus gewonnenen Erkenntnisse und Informationen. Diese Daten müssen dem Benutzer in entsprechenden Datenbanken zur Verfügung gestellt werden. In der Vergangenheit hat es sich leider gezeigt, dass dieser Prozess durch die damit verbundenen Kosten zum Ende eines Projektes führen kann. In dieser Arbeit sind verschiedene Strategien dargestellt worden, die zum Teil eine automatische Annotierung der Daten gestatten. Bei der Umsetzung sind zwei Erweiterungen der Sweet-Db entstanden. Die Algorithmen der Programme Glyco-Search-Ms und Glycan-Profiling gestatten eine schnelle Suche in einer theoretischen Vergleichsspektren-Sammlung. Bei der Verwaltung des Datenbestandes sind in erster Linie die Arbeitsumgebung zur Verwaltung von NMR- und Massenspektren zu nennen. Es wurde eine dezentrale Lösung geschaffen, die es dem Benutzer ermöglicht seine lokal gemessenen Spektren in dieser Datenbank zu verwalten. Hat er seine Ergebnisse veröffentlicht, können die Spektren über die beschriebenen Schnittstellen sofort in der Sweet-Db veröffentlicht werden. Dieses Vorgehen hat den Vorteil, dass die Daten ohne erneute Eingabe in die Datenbank übernommen werden können. In einem ersten Test wurden von zwei Hilfskräften ohne größere Probleme 347 Spektren über die Arbeitsumgebung eingeben und stehen nun der Sweet-Db zur Verfügung. Mit Hilfe der Programme autoReference und Reference konnte die Aktualisierung der Literatur zumindest semiautomatisch erfolgen. Ausgehend von einer Liste mit Trivialnamen kann in regelmäßigen Abständen in der Pubmed gesucht werden. Diese Rohdaten werden in einer temporären Datenbank zwischengespeichert und werden nach einer Kontrolle durch einen Experten in die Sweet-Db eingetragen

A New Method for Ligand-supported Homology Modelling of Protein Binding Sites: Development and Application to the neurokinin-1 receptor

In this thesis, a novel strategy (MOBILE (Modelling Binding Sites Including Ligand Information Explicitly)) was developed that models protein binding-sites simultaneously considering information about the binding mode of bioactive ligands during the homology modelling process. As a result, protein binding-site models of higher accuracy and relevance can be generated. Starting with the (crystal) structure of one or more template proteins, in the first step several preliminary homology models of the target protein are generated using the homology modelling program MODELLER. Ligands are then placed into these preliminary models using different strategies depending on the amount of experimental information about the binding mode of the ligands. (1.) If a ligand is known to bind to the target protein and the crystal structure of the protein-ligand complex with the related template protein is available, it can be assumed that the ligand binding modes are similar in the target and template protein. Accordingly, ligands are then transferred among these structures keeping their orientation as a restraint for the subsequent modelling process. (2.) If no complex crystal structure with the template is available, the ligand(s) can be placed into the template protein structure by docking, and the resulting orientation can then be used to restrain the following protein modelling process. Alternatively, (3.) in cases where knowledge about the binding mode cannot be inferred by the template protein, ligand docking is performed into an ensemble of homology models. The ligands are placed into a crude binding-site representation via docking into averaged property fields derived from knowledge-based potentials. Once the ligands are placed, a new set of homology models is generated. However, in this step, ligand information is considered as additional restraint in terms of the knowledge-based DrugScore protein-ligand atom pair potentials. Consulting a large ensemble of produced models exhibiting di erent side-chain rotamers for the binding-site residues, a composite picture is assembled considering the individually best scored rotamers with respect to the ligand. After a local force-field optimisation, the obtained binding-site models can be used for structure-based drug design

Myometrial cyclic AMP function

Background Uncovering the processes that drive labour onset is essential to reduce the adverse consequences of dysfunctional labour. Myometrial cAMP signalling is upregulated during pregnancy promoting uterine quiescence. Changes in its components and effectors have been identified at the onset of term labour. Preterm labour (PTL) treatments targeting this pathway have limited effectiveness and serious maternal effects. In this study, real-time FRET imaging was used to investigate compartmentalised cAMP signals at distinct cellular sites. Methods Myometrial biopsies were obtained from women at term or in distinct causes of PTL. Tissues were processed for mRNA and protein extraction or cell isolation. Primary myometrial cells (HPMCs) and an hTERT-HM cell line expressed either a cytosolic (EPAC-SH187) or plasmalemma (AKAP79-CUTie) genetically encoded FRET sensor. The florescence emission changes were monitored following isoproterenol and PGE2 treatment to determine intracellular cAMP concentrations. Results Differences in cAMP signalling components were detected in PTL compared to term with variations in effector predominance and an associated increase in OTR expression in twin-PTL. Stimulus-specific subcellular compartmentalisation of cAMP was identified in both cell types with differential regulation by phosphodiesterases (PDEs). Significant disparities were detected in the amplitude, kinetics, and regulation of cAMP signals between the two cell types. For the HPMCs, a prolonged time in culture was associated with a reduction in PDE activity and altered cell phenotype. Conclusion The cAMP signalling system is influential in the final pathway of labour, primarily regulating OTR expression. This study established the technique of FRET imaging in human myometrial cells, determining the cell model of choice and culture conditions to explore localised cAMP signalling. The findings provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium and pave the way for unravelling the details of how this fundamental pathway operates and its role in pregnancy and labour.Open Acces

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds