Design of thymidine-based inhibitors of Mycobacterium tuberculosis thymidylate kinase

Met de zoektocht naar krachtige en selectieve inhibitoren van het thymidine monophoshate kinase van Mycobacterium tuberculosis (TMPKmt), beschrijft dit werk de ontwikkeling van nieuwe anti-tuberculose geneesmiddelen. Aangezien TMPKmt een essentieel enzyme is voor de groei van de bacterie, kunnen inhibitoren van dit enzyme verder ontwikkeld worden tot anti-tuberculose geneesmiddelen. De X-stralen structuur van dit enzyme is bepaald in 2001 and toont de bindingswijze van het natuurlijke substraat dTMP en werd gebruikt om de activiteiten van gemodificeerde thymidine derivaten te verklaren en te voorspellen. Dit werk kadert in een eerder onderzoek in onze groep en bijgevolg kan de structuur-activiteitsrelatie die afgeleid werd uit vroeger gesynthesiseerde componenten gebruikt worden voor verdere ontwikkeling van nieuwe thymidine derivaten. De meest interessante topics van de reeds gekende structuur-activiteitsrelatie hebben gediend als startpunt van dit werk

Protein-Ligand Interactions: Target Identification and Drug Discovery

Bioactive compounds and drugs are designed and screened on the basis of specific molecular targets as well as via the identification of active ingredients from traditional medicine or by serendipitous discovery. The development of novel therapeutic strategies not only requires a deep knowledge of the molecular processes and the cellular pathways involved in each pathological condition and disease, but also the specific protein targets and the effects of drug binding on protein conformation and activity. Understanding of how drugs can modify and modulate specific cellular pathways and functions will be helpful during the process of drug development and clinical trials

Towards intelligent drug design system: Application of artificial dipeptide receptor library in QSAR-oriented studies

The pharmacophore properties of a new series of potential purinoreceptor (P2X) inhibitors determined using a coupled neural network and the partial least squares method with iterative variable elimination (IVE-PLS) are presented in a ligand-based comparative study of the molecular surface by comparative molecular surface analysis (CoMSA). Moreover, we focused on the interpretation of noticeable variations in the potential selectiveness of interactions of individual inhibitor-receptors due to their physicochemical properties; therefore, the library of artificial dipeptide receptors (ADP) was designed and examined. The resulting library response to individual inhibitors was arranged in the array, preprocessed and transformed by the principal component analysis (PCA) and PLS procedures. A dominant absolute contribution to PC1 of the Glu attached to heptanoic gating acid and Phe bonded to the linker m-phenylenediamine/triazine scaffold was revealed by the PCA. The IVE-PLS procedure indicated the receptor systems with predominant Pro bonded to the linker and Glu, Gln, Cys and Val directly attached to the gating acid. The proposed comprehensive ligand-based and simplified structure-based methodology allows the in-depth study of the performance of peptide receptors against the tested set of compounds.NC

Rum aroma descriptive analysis

Rum contains a large number of compounds that contribute to the complex aroma, some of which present in a very small amount. This fact represents a problem for the use of chemical analysis as a way to ascertain the quality of rum and to determine differences in rums products. Descriptive sensory analysis techniques can be useful in such circumstance, not only because they are easier to achieve, but because they give valuable information on the actual perceived aroma of the products. The first part of this work consisted of the development of lexicon for describing rums. A group of 5 semi-expert judges evaluated a variety of 15 commercial rums from different origins, raw materials and processing conditions, and created consensually a list of 33 terms with description useful for describing the aroma, flavor, and taste of rums. The second part of the work was focused on the creation of a method used to describe the aroma of different rum products, to discriminate among different rums, and to relate the perceived aroma of the samples prepared from different processing protocols. Using a group of 12 trained panelists, a modified descriptive analysis technique for the evaluation of rum aroma, and the adequate descriptors and references that were generated, it was possible to describe and discriminate rum samples. The results obtained from the evaluation of the 9 different commercial rums and one experimental sample can be used to relate the perceived aroma of the different rum samples to their processing protocol. Among the many possible applications of this study is the use as a tool for quality control, new product development, and brand identification. The information obtained from the description of the products can also be a useful tool for marketing purposes

Effects of glycation by methylglyoxal in saccharomyces cerevisae

Mestrado em Bioquímica (Bioquímica Médica) Universidade de Lisboa; Faculdade de Ciências, 2021Glycation is the nonenzymatic modification of biomolecules caused by reactive dicarbonyl species. In vivo, the major physiological glycation agent is methylglyoxal. This compound’s formation occurs in all living cells, mainly nonenzymatically from dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate during glycolysis. When glycation occurs on proteins it originates advanced glycation end-products (AGEs), which will change the structure and stability of proteins, consequently impairing their functionality. AGEs tend to accumulate on cells, being associated with ageing and several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer’s disease, Parkinson’s disease and transthyretin amyloidosis). Hence, there is a growing interest in this post-translational modification. However, little is known about the physiologic effects of glycation, since most studies use in vitro approaches, which do not fully reproduce the in vivo process. Saccharomyces cerevisiae is an ideal model of in vivo glycation, being able to reproduce the effects that happen in higher eukaryotic cells. The in vivo study and use of Saccharomyces cerevisiae cells with different genetic backgrounds, gives us an accurate vision of a wide range of phenotypical and physiological effects caused by glycation. In the present study we show that the glyoxalase system seems to be the major detoxifying mechanism of methylglyoxal, since mutations on this pathway lead to lower growth rates. Moreover, using fluorescence microscopy and flow cytometry we found that glycation is not sufficient to alter cell morphology or its viability. However, the effects of glycation seem to be devastating on mitochondrial activity causing its dysfunction and the accumulation of reactive oxygen species (ROS). These findings provide insight to uncover knowledge of the process of glycation, necessary for better comprehension and advance in the cure and early identification of many human diseases that haunt our society

Characterization and exploitation of protein ligand interactions for structure based drug design

Most characterised protein-small molecule interactions that display a change in heat capacity (\bigtriangleupCp) occur with a negative \bigtriangleupCp value. This is often attributed to solvent reorganisation from reduction in solvent accessible apolar surface area accompanying complex formation. Positive \bigtriangleupCp values have not been widely reported and could typically be attributed to an increased solvent accessible apolar surface area, desolvation of polar surface area or structural transitions in the biomolecular complex. Heat shock protein-90 (Hsp90) is one of the abundant and important molecular ATP-dependent chaperones. The N-terminal domain of Hsp90 contains ATP/ADP binding site, where Hsp90-ADP interactions proceed with a large positive \bigtriangleupCp of 2.35 ± 0.46 kJ·mol-1·K-1. Interestingly geldanamycin, an Hsp90 inhibitor which binds to the same N-Hsp90-ADP/ATP binding site, interacts with a negative \bigtriangleupCp of -0.39 ± 0.04 kJ·mol-1·K-1. The semi-empirical correlation of the solvent accessible surface area change does not match well with the observed \bigtriangleupCp. This prompted us to investigate various factors affecting the thermodynamics of protein-small molecule binding including varying buffers, differing salt concentration, altering pH, substitution of different metal cations and performing interactions in heavy water. Molecular dynamics simulation and NMR studies have allowed us to disregard structural changes of N-Hsp90-ADP molecule from giving rise to positive \bigtriangleupCp. From a combination of these calorimetric, simulation and structural studies we have gathered a considerable body of evidence suggesting that the change in accessible surface area, ionic interactions and resultant desolvation of water molecules from the surface of a Mg2+ ion can contribute substantially to a positive \bigtriangleupCp. We conclude that this unique result appears to come from extensive disruption of the tightly bound water molecules present around Mg2+-ADP after binding to Hsp90, which then gives rise to a positive \bigtriangleupCp. In addition to these findings, the thermodynamics of 18 structurally related CDK2 inhibitors were investigated using ITC. CDK2 is a member of cyclin dependent kinases implicated in eukaryotic cell cycle progression and control. This investigation showed that even conservative changes in small molecule structure can reveal large variation in thermodynamic signature, while simple concepts such as van der Waals interactions, steric hindrance, and hydrophobicity are insufficient to explain it

Design, Synthesis, Characterization and Biological Evaluation of Some Novel Thiadiazole (Schiff’s Base) Derivatives as Antitubercular Agents against Glutamine Synthetase I

Glutamine synthetase I is a vital enzyme present in the cell wall of Mycobacterium tuberculosis H37Rv. It belongs to the Ligase family. • A database of 200 molecules with high prospects of inhibiting the target Glutamine synthetase I were carefully chosen by making changes to the known hit molecules, here the thiadiazole nucleus was chosen. • The designed molecules were docked against the target chosen using AutoDock 4®. • Five molecules with good docking score [lower binding energy] and interactions were shortlisted for synthesis. • The selected molecules were subjected to toxicity prediction assessment by OSIRIS® property explorer developed by Acetilon Pharmaceuticals limited which is available online. The results are color coded as green color which predicts the drug likeness and possibly better activity. • The reaction condition were optimized, synthesized and labelled as BOB, CIN, NIB, OHB, PHB. • The characterization of the synthesized compounds was done using TLC, Melting point Infra-red, Mass spectrometric methods [LC-MS] and Nuclear Magnetic Resonance [H1 NMR] spectroscopy methods. • All the Synthesized compounds exhibited molecular ion peak (M+) of varying intensities. • The final pure compounds were screened for Anti-mycobacterial activity by in vitro method called Microplate Alamar Blue Assay [MABA]. • The synthesized compounds showed sensitivity [Minimum inhibitory concentration] at 3.125mcg/ml. The standard drugs Pyrazinamide, Ciprofloxacin and Streptomycin exhibited anti-mycobacterial activity at 3.125 mcg/ml, 3.125 mcg/ml and 6.25 mcg/ml concentrations respectively. This indicates that the synthesized compounds are as Potent as the standard drugs. • Based on the MABA report, Acute Oral Toxicity study were performed and observed that the administration of the synthesized molecules by oral route upto 2000mg/kg/b.w is safe. • The selected compounds showed IC50 Values of 121.5, 201.9, 472.9, 456.2 μg/ml respectively for BOB, NIB, OHB, PHB. Rifampicin showed IC50 value of 113 μg/ml. As compared with standard drug the synthesized molecules were found to be more cytotoxic. CONCLUSION: It is concluded that the synthesized compounds might effectively inhibit the chosen target Glutamine Synthetase I which is essential for the Mycobacterial tuberculosis. Further structural modifications of the synthesized compounds will aid in the development of potential molecule against the pathogen

Tartu Ülikooli keemiaosakond 1947-2002

http://www.ester.ee/record=b1720730*es

An insight towards food-related microbial sets through metabolic modelling and functional analysis

The dietary food digestion depends on the human gastrointestinal tract, where host cells and gut microbes mutually interact. This interplay may also mediate host metabolism, as shown by microbial-derived secondary bile acids, needed for receptor signalling. Microbes are also crucial in the production of fermented foods, such as wine and dairy. Kefir is fermented milk processed by the symbiotic community of bacteria and yeasts. One such species is a yeast Kluyveromyces marxianus. Its thermotolerance is a desired trait in biotechnology since it may reduce the cooling demands during cultivation.The systems biology tools allow analysing various size microbial communities under the different functional scope. For example, the homology prediction tools can give detailed functional insights when working with metagenomics data. The whole-cell metabolic processes can be summarised in genome-scale metabolic models (GEMs), which enable to predict the metabolic capabilities and allow for the integration of omics data.The work shown in this thesis includes i) in silico analysis of food-related microbes; ii) the development of GEMs and RAVEN. With a focus on bile acid metabolism, hundreds of human gut microbes were annotated based on metagenomics data, thereby suggesting the differences in the potential for bile acid processing between healthy and diseased subjects. These findings may be exploitable once aiming to restore the bile acid metabolism for the patients having inflammatory bowel disease. Also, the metabolism of yeast K. marxianus was characterised in genome-scale. Two K. marxianus strains from kefir grains were isolated, sequenced, assembled, and functionally annotated. They were compared with the other ten strains, providing the core and dispensable physiological features for K. marxianus. Furthermore, the first GEM for K. marxianus, namely iSM996, was reconstructed. It was integrated with transcriptomics data to predict its metabolic capabilities in rich medium and high-temperature conditions. The results might be useful to optimise strain-specific medium for high-temperature applications. The final paper comprises the efforts to improve the usability for RAVEN, a toolbox for GEM reconstruction and analysis. Altogether the outcomes of this thesis suggest the potential applications for medicine and industrial biotechnology, which may be facilitated by the newly upgraded RAVEN toolbox