The aim of the study was to analyse where different organic molecules situated themselves in relation to the water surface of a water microdroplet and use the resulting data to compare three different forcefields in the simulation package GROMACS. The forcefields used were: General AMBER forcefield (GAFF), Optimized potentials for liquid simulations - all atoms (OPLS-AA), and CHARMM general force field (CGenFF). A library of 146 molecules were simulated using molecular dynamics. Out of the 146 molecules only 65 resulted in useful data for the comparison of the forcefields. The molecules were placed in the centre of a water microdroplet and their movements were simulated for a duration of 1 ns. The trajectories and positions of the molecules were stored and from each simulation a density profile was generated, showing where the molecules situated themselves. The distance from the peak of the density profile to the water surface was calculated and compared between the different forcefields. To analyse the data further some of the molecules were divided into subsets based on their functional groups to see if any trends were visible. Although inconclusive, the data suggested that different forcefields were more or less agreeable depending on the functional group of the molecules, for example OPLS-AA differed from CGenFF and GAFF in the case of alcohols.

La résistance aux antibiotiques est une menace sérieuse pour la santé publique. En effet, si on ne change pas rapidement notre consommation excessive d'antibiotiques, la situation actuelle va se dégrader jusqu'à basculer dans une ère dite "post-antibiotique", dans laquelle plus aucun antibiotique ne sera efficace contre les infections microbiennes. Bien que ce phénomène de résistance apparaît naturellement, l'utilisation abusive d'antibiotiques accélère le processus. De plus, la présence de pathogènes multi-résistants neutralise l'effet des traitements existants et dans le cas de chirurgies courantes (césariennes, transplantations d'organe...), la situation peut rapidement s'aggraver voire devenir mortelle. C'est pourquoi des directives, émanant des autorités sanitaires, doivent être mises en place afin de contrôler l'utilisation des médicaments, et ce, à tous les niveaux de la société, des individus au secteur agricole en passant par les professionnels de santé et les industries pharmaceutiques. Le monde de la recherche scientifique, quant à elle, doit trouver des nouvelles stratégies pour enrayer la propagation de la résistance. Dans ce contexte, cette thèse a pour objectif le développement d'une méthode de prédiction, par calculs d'énergie libre, des mutations de β-lactamases favorables à l'hydrolyse des β-lactames. Ces travaux méthodologiques ont donc conduit au développement : (1) de nouveaux paramètres pour les enzymes à zinc, implémentés dans le champ de force OPLS-AA et validés par des simulations de dynamique moléculaire sur un panel de métalloenzymes représentatives, (2) d'un protocole de paramétrisation de ligands covalents pour étudier le comportement de certains β-lactames dans CMY-136, une nouvelle β-lactamase caractérisée au laboratoire, et (3) d'un protocole de calcul d'énergie libre évalué au moyen de compétitions internationales de prédiction. Ce dernier a ensuite été utilisé pour tenter d'expliquer pourquoi la carbamylation de la sérine catalytique n'a pas lieu dans certaines oxacillinases. Au travers de ces travaux, nous avons pu améliorer significativement notre approche computationnelle et désormais tout est en place pour une exploration exhaustive des mutations possibles dans les β-lactamases. / Antibiotic resistance is a global concern threatening worldwide health. Indeed, if we don't change our overconsumption of antibiotics, the current situation could worsen until a "post-antibiotic" era in which existing treatment would be ineffective against microbial infections. Despite the natural occurrence of antibiotic resistance, the misuse of antibiotics is speeding up the process. Furthermore, presence of multi-resistant pathogens negates the effect of modern treatments and usual surgeries (caesarean sections, organ transplantations...) might be riskier in the future, or even lethal. That's why, common guidelines have to be edicted by health authorities in order to control antibiotic use at every level of society, from individuals to healthcare industry including health professionals and agriculture sector. As for scientific research, new strategies have to be considered in order to limit the spread of antibiotic resistance. In that context, the presented thesis aimed at developing a protocol to predict, by free energy calculations, β-lactamase mutations which could promote the hydolysis of β-lactams antibiotics. In order to achieve that, we developed several methodological approaches including: (1) new parameters for zinc enzymes implemented in OPLS-AA force field and thereafter validated using molecular dynamics simulations of representative zinc-containing metalloenzymes, (2) a protocol to parameterize covalent ligands in order to analyze the dynamical behavior of some β-lactams in CMY-136, a novel β-lactamase recently characterized in our laboratory, and (3) a pmx-based free energy protocol. The latter was also assessed through several international blinded prediction challenges, and finally used to find out why carbamylation of the catalytic serine is not observed in certain OXA enzymes. Throughout this work, we made significant improvements in our protocol, and now everything is in place for an exhaustive prediction of possible mutations in β-lactamases.

Project I Celogentin C, isolated by Kobayashi from the seeds of Celosia argentea in 2001 exhibits strong inhibitory behavior towards polymerization of tubulin – globular protein, which plays crucial role during the cell division cycle. We have attempted synthesis of the left-hand ring of celogentin C via the intramolecular alkylation and the Knoevenagel condensation strategies. Utilizing synthetic methodologies in the field of nitroacetyl chemistry, developed by Kornblum and Rajappa we have successfully prepared the intermolecular Knoevenagel condensation product – the late-stage precursor to the left-hand ring of celogentin C. Synthesis of this key intermediate subsequently led to the preparation of the left-hand ring and the total synthesis of celogentin C by other members of Castle's group. Project II Thioviridamide is the potent apoptosis inducer isolated by Hayakawa from the bacterial broth of Streptomyces olivoviridis. Unusual structural features of the thioviridamide macrocycle contain two novel amino acids, never before encountered in a natural product - S-(2-aminovinyl)cysteine (avCys) and β-hydroxy-N1,N3-dimethylhistidinium (hdmHis). No stereochemical information except for the Z-configuration of the β-thioenamide linkage was reported in the literature. We have performed a computational study to predict the thioviridamide stereochemistry. Initial populations of conformers for the likely candidate structures were produced using OPLS-AA force field. Prediction of the NMR properties was accomplished at the mPW1PW91/6-311+G(2d,p) level of theory with the polarizable continuum model of salvation. Utilizing Boltzmann averaging and statistical analysis we have determined that the only possible cases of stereochemical inversion occur at the sites of the two novel amino acids. Project III Model studies towards the synthesis of the β-thioenamide subunit of thioviridamide were performed. The radical addition reaction of thiyl radicals to ynamides produces Z- (kinetic products) or E- β-thioenamides (thermodynamic products) depending on the reaction conditions. Two distinct sets of reaction conditions allowing kinetic or thermodynamic control of β-thioenamide formation were developed. Synthesis of the model β-thioenamide subunit of thioviridamide was attempted.