No / In this paper, molecular modelling was used to investigate the nature of probe/surface interactions during the analysis of Dß-mannitol using inverse gas chromatography (IGC). IGC was used to experimentally measure the dispersive components of surface free energy () and the specific components of free energy of adsorption () of Dß-mannitol by calculating the retention time of non-polar (n-alkanes) and polar (tetrahydrofuran and chloroform) probes, respectively. The results showed that Dß-mannitol surface is acidic in nature because the basic probe had more interaction with the surface as compared to acidic probe. Cerius2 software package was used to model the two morphologically important surfaces, which showed the presence of surface hydroxyl groups. Molecular dynamics simulations were performed in Cerius2 to model the adsorption of the same probes (n-alkanes, tetrahydrofuran and chloroform) on the Dß-mannitol surfaces. The adsorption energies calculated from the simulation showed a close match to those determined experimentally. The calculated values are slightly higher for all probes except chloroform, but as a single perfect crystal was modelled without considering the effect of impurities, solvent and other physical factors this is not unexpected.

No / The surface energy of a-lactose monohydrate measured by inverse gas chromatography (IGC) is reported along with a dynamic molecular modelling study of the interaction of the various molecular probes with different surfaces of a-lactose monohydrate. The IGC results show that a-lactose monohydrate is acidic in nature. Using quantitative calculations of the energy of adsorption, the acidic nature of the surface is confirmed and the calculated values agree closely with the experimentally measured values. Along with the acidic nature, dynamic molecular modelling also reveals that the presence of a channel and water molecules on a surface affects the surface energetics of that face. The presence of water on the surface can decrease or increase the surface energy by either blocking or attracting a probe molecule, respectively. This property of water depends on its position and association with other functional groups present on the surface. The effect of a channel or cavity on the surface energy is shown to depend on its size, which determines whether the functional groups in the channel are assessable by probe molecules or not. Overall molecular modelling explains, at the molecular level, the effect of different factors affecting the surface energy of individual faces of the crystal.

Yes / A new type of model, FFLUX, to describe the interaction between atoms has been developed as an alternative to traditional force fields. FFLUX models are constructed from applying the kriging machine learning method to the topological energy partitioning method, Interacting Quantum Atoms (IQA). The effect of varying parameters in the construction of the FFLUX models is analyzed, with the most dominant effects found to be the structure of the molecule and the number of conformations used to build the model. Using these models the optimization of a variety of small organic molecules is performed, with sub kJ mol-1 accuracy in the energy of the optimized molecules. The FFLUX models are also evaluated in terms of their performance in describing the potential energy surfaces (PESs) associated with specific degrees of freedoms within molecules. While the accurate description of PESs presents greater challenges than individual minima, FFLUX models are able to achieve errors of <2.5 kJ mol-1 across the full C-C-C-C dihedral PES of n-butane, indicating the future possibilities of the technique.

Basische Benzilsäureester stellen mit ihrer ausgeprägten anticholinergen Wirksamkeit potenzielle Arzneistoffe zur Behandlung der Harninkontinenz, der Ulkuserkrankung und des Morbus Parkinson dar. Von besonderem Interesse sind Benzilsäurevertreter, die neben anticholinergen auch dopaminerge Effekte aufweisen. Wegen ihrer dualistischen Wirkung könnten sie eine neue Klasse von Antiparkinsonica begründen. Aufgrund der vielfältigen Funktionen von Muscarinrezeptoren treten bei wenig selektiv wirksamen Arzneistoffen atropinartige Nebenwirkungen auf. Mit der Entwicklung von Verbindungen, die eine erhöhte muscarinerge Subtypenselektivität besitzen, lassen sich Nebenwirkungen reduzieren. Ziel der Arbeit war eine Wirkungsoptimierung chiraler N-Methyl-4-piperidyl benzilate durch Variation von stereochemischen Parametern und Einführen elektronisch verschiedenartiger Substituenten in die aromatischen Ringe. In Radioligand-Bindungsstudien an M1- bis M3-Rezeptoren wurden die Auswirkungen der sterischen und strukturellen Variationen untersucht. Die Ergebnisse der Bindungsstudien zeigen, dass sich Affinität und Subtypenselektivität durch die absolute Konfiguration des stereogenen Zentrums und die Art der Kernsubstitution modifizieren lassen. Mit Hilfe von Molecular Modelling ist es gelungen, auf Basis der experimentellen Bindungsdaten ein aussagekräftiges Rezeptormodell für N-Methyl-4-piperidyl benzilate zu entwickeln. Sowohl die Affinitätsunterschiede enantiomerer Benzilate als auch die Unterschiede der Rezeptorsubtypenselektivität werden durch das Rezeptormodell umfassend erklärt. / Basic substituted benzilic esters with distinctive anticholinergic effects are potential drugs for the treatment of urinary incontinence, duodenal and gastric ulcers and Parkinson disease. Derivatives of benzilic esters, exhibiting a combination of anticholinergic and dopaminergic effects, are of special interest because, as a consequence of their dualistic effect, they are in a position to form a new class of Antiparkinson drugs. As muscarinic receptor subtypes possess a large variety of functional properties, drugs which show less selectivity on muscarinic receptors exhibit atropine-like side effects. A reduction of these side effects may be achieved by the development of more selective anticholinergic compounds. The objective was to optimise the effect of N-Methyl-4-piperidyl benzilates through a variation of sterical parameters and the introduction of electronically differentiated substituents within the aromatic rings. The effect of sterical and structural variations was investigated in radioligand binding studies on muscarinic receptors (M1 – M3). The results of these binding studies reveal that a modification of affinity and selectivity can be achieved by varying the absolute configuration of the stereogenic center and the properties of the substitution of the aromatic system. The development of a relevant model of the receptor ligand complex for N-Methyl-4-piperidyl benzilates was achieved by molecular modelling on the basis of experimental binding studies. Both the diverse affinity of enantiomeric benzilic esters and the subtype selectivity on muscarinic receptors are comprehensively explained by this model.

The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC–MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

Micronisation of active pharmaceutical ingredients (APIs) to achieve desirable quality attributes for formulation preparation and drug delivery remains a major challenge in the pharmaceutical sciences. It is therefore important that the relationships between crystal structure, the mechanical properties of powders and their subsequent influence on processing behaviour are well understood. The aim of this project was therefore to determine the relative importance of particle attributes including size, crystal quality and morphology on processing behaviour and the characteristics of micronised materials. It was then subsequently intended to link this behaviour back to crystal structure and the nature of molecular packing and intermolecular interactions within the crystal lattice enabling the identification of some generic rules which govern the quality of size reduced powders. In this regard, different sieve fractions of lactose monohydrate and crystal variants of ibuprofen and salbutamol sulphate (size, morphology and crystal quality) were investigated in order to determine those factors with greatest impact on post-micronisation measures of particle quality including particle size, degree of crystallinity and surface energy. The results showed that smaller sized feedstock should typically be used to achieve ultrafine powders with high crystallinity. This finding is attributed to the reduced number of fracture events necessary to reduce the size of the particles leading to decreases in milling residence time. However the frequency of crystal cracks is also important, with these imperfections being implicated in crack propagation and brittle fracture. Ibuprofen crystals with a greater number of cracks showed a greater propensity for comminution. Salbutamol sulphate with a high degree of crystal dislocations however gave highly energetic powders, with reduced degree of crystallinity owing to the role dislocations play in facilitating plastic deformation, minimising fragmentation and extending the residence of particles in the microniser. Throughout these studies, morphology was also shown to be critical, with needle like morphology giving increased propensity for size reduction for both ibuprofen and salbutamol sulphate, which is related to the small crack propagation length of these crystals. This behaviour is also attributed to differences in the relative facet areas for the different morphologies of particles, with associated alternative deformation behaviour and slip direction influencing the size reduction process. Molecular modelling demonstrated a general relationship between low energy slip planes, d-spacing and brittleness for a range of materials, with finer particle size distributions achieved for APIs with low value of highest d-spacings for identified slip planes. The highest d-spacing for any material can be readily determined by PXRD (powder x-ray diffraction) which can potentially be used to rank the milling behaviour of pharmaceutical materials and provides a rapid assessment tool to aid process and formulation design. These studies have shown that a range of crystal properties of feedstock can be controlled in order to provide micronised powders with desirable attributes. These include the size, morphology and the density of defects and dislocations in the crystals of the feedstock. Further studies are however required to identify strategies to ensure inter-batch consistency in these attributes following crystallisation of organic molecules.

The rationalisation of drug potency using three-dimensional structures of protein-ligand complexes is a central paradigm in medicinal research. For over two decades, a major goal has been to find the rules that accurately relate the structure of any protein-ligand complex to its affinity. Addressing this problem is of great concern to the pharmaceutical industry, which uses virtual screens to computationally assay up to many millions of compounds against a protein target. A fast and trustworthy affinity estimator could potentially streamline the drug discovery process, reducing reliance on expensive wet lab experiments, speeding up the discovery of new hits and aiding lead optimization. Water plays a critical role in drug-protein interactions. To address the often ambiguous nature of water in binding sites, a water placement method was developed and found to be in good agreement with X-ray crystallography, neutron diffraction data and molecular dynamics simulations. The method is fast and has facilitated a large scale study of the statistics of water in ligand binding sites, as well as the creation of models pertaining to water binding free energies and displacement propensities, which are of particular interest to medicinal chemistry. Structure-based scoring functions employing the explicit water models were developed. Surprisingly, these attempts were no more accurate than the current state of the art, and the models suffered from the same inadequacies which have plagued all previous scoring functions. This suggests a unifying cause behind scoring function inaccuracy. Accordingly, mathematical analyses on the fundamental uncertainties in structure-based modelling were conducted. Using statistical learning theory and information theory, the existence of inherent errors in empirical scoring functions was proven. Among other results, it was found that even the very best generalised structure-based model is significantly limited in its accuracy, and protein-specific models are always likely to be better. The theoretical framework developed herein hints at modelling strategies that operate at the leading edge of achievable accuracy.

Molecular modelling has become a well-established tool for studying biological mole- cules, moreover with the prospect of being useful for drug development. The thesis summarises research on the methodological advances in the treatment of molecular flexibility and intermolecular interactions. Altogether, seven original publications are accompanied by a text which aims to provide a general introduction to the topic as well as to emphasise some consequences of the computer-aided drug design. The molecular flexibility is tackled by a study of a drug-DNA interaction and also by an investigation of small drug molecules in the context of implicit solvent models. The approaches which neglect the conformational freedom are probed and compared with experiment in order to suggest later, how to cope with such a freedom if in- evitable. The noncovalent interactions involving halogen atoms and their importance for drug development are briefly introduced. Finally, a model for a faithful description of halogen bonds in the framework of molecular mechanics is developed and its per- formance and limits are tested by a comparison with benchmark ab initio calculations and experimental data. 1

Os inibidores do receptor de angiotensina II do tipo 1 (AT1R), fármacos da classe das sartanas, são muito utilizados na terapêutica da insuficiência cardíaca. Apesar de serem eficientes por baixarem a pressão arterial, esses inibidores diminuem a contratilidade do músculo cardíaco, acentuando a patologia. Nesse sentido, os agonistas enviesados para &#946;-arrestina do AT1R surgem como uma solução para esse problema. Estudos com o mais promissor peptídeo com ação agonista enviesada (TRV120027) mostram que ele é capaz de diminuir a pressão arterial sem causar o efeito inotrópico negativo no coração. Tendo em vista esse novo e promissor mecanismo de ação e a característica peptídica do novo agonista enviesado que restringe sua utilização, o presente trabalho visou à busca de ligantes não peptídicos com potencial ação enviesada. Foram realizados estudos de ancoramento seguidos de dinâmica molecular, no AT1R, de sete peptídeos agonistas e agonistas enviesados descritos na literatura, empregando-se os programas Surflex-Dock 2.0 e o GROMACS 4.5, além de análises de campos de interação molecular no programa GRID. Os dados das interações intermoleculares retirados da dinâmica e dos campos de interação guiaram a construção de um farmacóforo que foi utilizado posteriormente em uma busca virtual na base de dados ZINC, com o módulo UNITY 3D do pacote Sybyl-X Suite 2.0. Após ancoramento e análise visual das moléculas selecionadas na busca, foram identificadas 15 moléculas promissoras, sendo cinco delas consideradas de maior interesse. As moléculas selecionadas na busca poderão ser futuramente testadas quanto ao perfil de ação enviesada em receptores AT1R. Os resultados obtidos nesse estudo podem levar à descoberta de um novo protótipo mais eficiente e seguro para o tratamento de doenças cardiovasculares, como a insuficiência cardíaca. / Angiotensin II type 1 receptor (AT1R) inhibitors, the sartans, are widely used in the treatment of heart failure. Although they are effective for lowering blood pressure, these inhibitors decrease the contractility of the heart muscle, accentuating the pathology. Accordingly, &#946;-arrestin biased agonists for AT1R emerge as a solution to this problem. Studies with the most promising biased agonist peptide (TRV120027) show that it is able to lower blood pressure without causing negative inotropic effect on the heart. Given this promising new mechanism of action and the peptide feature of the new agonist that restricts its use, this work aims the search for non-peptide ligands with a potential biased action. Docking studies, followed by molecular dynamics simultions, were performed for seven full and biased agonists in the AT1R, using the Surflex-Dock 2.0 and 4.5 GROMACS programs, besides molecular interaction fields analysis with GRID software. The data of intermolecular interactions from the molecular dynamic\'s analysis and the molecular interaction fields guided to the construction of a pharmacophore model which was subsequently used in a virtual screening from ZINC database, employing the 3D UNITY module from Sybyl-X Suite 2.0 package. After a docking study and visual analysis of the primary selected molecules, 15 promising molecules have been identified, five of them considered of most interest. The molecules selected in the search can be further tested for biased action on AT1R. The results of this study may lead to the discovery of a more efficient and secure lead for the treatment of cardiovascular diseases, such as heart failure.

O metabolismo de vitamina B9 é um alvo terapêutico conhecido para malária. A vitamina B6 foi validada como essencial para o parasita. Esse trabalho analisa bioquimica- e estruturalmente mutantes da enzima piridoxal quinase, pela combinação de análises in silico associadas a ensaios bioquímicos. A estrutura da PdxK de P. falciparum permitiu a localização do sítio ativo e da interface de dimerização. Logo foram sugeridas mutações que alterassem esses resíduos para investigar sua importância. Dinâmica molecular sugere que a dimerização é responsável pela estabilidade do sítio ativo, algo coerente com a diminuição da atividade enzimática na maioria das mutantes. Filtração em gel aponta um equilíbrio entre a conformação monômero-dímero mostrando que as mutações na interface não estão relacionadas a dimerização, mas envolvidas com a estabilização do folding na região do sítio ativo. A mesma é recoberta por uma tampa que impede a auto-hidrólise do ATP, há também um resíduo serina que estabiliza a conformação do piridoxal durante a catálise, as mutações em ambas as regiões levaram a inativação da enzimática. A hipótese de que a interação da PfPdxK com ligantes de RNA não se mostrou conclusiva. / Vitamin B9 metabolism is a known drug-target for malaria. Vitamin B6 was validated as essential for the parasite. We analysed biochemically and structurally the plasmodial dimeric pyridoxal kinase. PfPdxKs allowed us to determine the localisation of the active site as well the interface between the two monomers and to identify the involved residues. Molecular dynamics shows that the PdxKs dimerization is important for the active sites stability, which was confirmed by the decrease of activity in mutants related to this region. Gel filtration revealed equilibrium of monomer-dimer conformation and therefore the interface mutations decrease in activity might not be directly related to the dimerization processes, but rather to the active site organisation. The active site region shows a serine involved in keeping the pyridoxals conformation during catalyses and the identified lid region covering the ATP binding site is responsible for preventing auto-hydrolysis. Substitution of the respective amino acid to alanine resulted in enzyme inactivation. In silico analysis of the PfPdxK spacer region identified nucleic acid binding sides, however RNA binding experiments failed so far and the possibility of protein-RNA binding remains for elucidation.