Synthèse d’inhibiteurs potentiels non phosphorylés de la désoxyxylulose phosphate réductoisomérase et étude de la voie de biosynthèse des unités isopréniques chez Acanthamoeba polyphaga. De nombreux microorganismes pathogènes utilisent la voie du méthylérythritol phosphate (MEP) pour la synthèse des unités isopréniques (IPP et DMAPP). Absente chez l’homme, cette voie constitue une cible de choix pour lutter contre ces microorganismes. La fosmidomycine est un des meilleurs inhibiteurs connus à ce jour de la désoxyxylulose phosphate réductoisomérase (DXR), deuxième enzyme de cette voie. Afin d’améliorer d’une part la biodisponibilité de ce type d’inhibiteur et d’autre part de lutter contre le phénomène de résistance à cet antibiotique, nous avons synthétisé des analogues de la fosmidomycine où le groupement phosphonate est remplacé par un groupement tétrazole ou par un groupement squaryle. Les molécules synthétisées ont été testées sur la DXR d’ Escherichia coli et n’ont pas révélé d’activité inhibitrice significative.Par ailleurs, nous avons montré, par des expériences d’incorporation de glucose marqué au 13C, que l’amibe Acanthamoeba polyphaga, utilise la voie du mévalonate pour synthétiser les unités isopréniques nécessaires à la synthèse de ses stérols / Synthesis of potential non-phosphonate inhibitors of the deoxyxylulose phosphate reductoisomerase and study of the biosynthetic pathway for isoprenoids units synthesis using by Acanthamoeba polyphagaMany pathogenic microorganisms synthesize their isoprenoid units (IPP and DMAPP) via the methylerythritol phosphate pathway (MEP pathway). Absent in man, all enzymes of this metabolic route are potential targets for the design of new antimicrobials. This pathway is present in pathogenic bacteria, but absent in mammals. Hence, the development of small-molecule inhibitors for the MEP enzymes constitutes a novel approach for the design of new antimicrobials. Fosmidomycin is the most efficient inhibitor of the the deoxyxylulose phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. In an attempt to improve the pharmacological properties and the bioavailability of this antibiotic, we synthesized analogues of the fosmidomycin by replacing the phosphonate group by tetrazole or squaryl moieties. These synthesized compounds were tested on the DXR isolated from Escherichia coli.Otherwise, we showed by achieving incorporation experiments with 13C labeled glucose that the amoeba Acanthamoeba polyphaga utilize the mevalonate pathway to synthesize its sterols.

The recent emergence of a number of new viral diseases as well as the re-emergence of tuberculosis (TB), indicate an urgent need for new drugs against viral and bacterial infections. Coronavirus nsp1 has been shown to induce suppression of host gene expression and interfere with host immune response. However, the mechanism behind this is currently unknown. Here we present the first nsp1 structure from an alphacoronavirus, Transmissible gastroenteritis virus (TGEV) nsp1. Contrary to previous speculation, the TGEV nsp1 structure clearly shows that alpha- and betacoronavirus nsp1s have a common evolutionary origin. However, differences in conservation, shape and surface electrostatics indicate that the mechanism for nsp1-induced suppression of host mRNA translation is likely to be different in the alpha- and betacoronavirus genera. The Modoc virus is a neuroinvasive rodent virus with similar pathology as flavivirus encephalitis in humans. The flaviviral methyltransferase catalyses the two methylations required to complete 5´ mRNA capping, essential for mRNA stability and translation. The structure of the Modoc NS5 methyltransferase domain was determined in complex with its cofactor S-adenosyl-L-methionine. The observed methyltransferase conservation between Modoc and other flaviviral branches, indicates that it may be possible to identify drugs that target a range of flaviviruses and supports the use of Modoc virus as a model for general flaviviral studies. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is part of the methylerythritol phosphate (MEP) pathway that produces essential precursors for isoprenoid biosynthesis. This pathway is used by a number of pathogens, including Mycobacterium tuberculosis and Plasmodium falciparum, but it is not present in humans. Using a structure-based approach, we designed a number of MtDXR inhibitors, including a novel fosmidomycin-analogue that exhibited improved activity against P.falciparum in an in vitro blood cell growth assay. The approach also allowed the first design of an inhibitor that bridge both DXR substrate and co-factor binding sites, providing a stepping-stone for further optimization.

Since the discovery of Mycobacterium tuberculosis (Mtb) as the bacterial agent causing tuberculosis, the permanent eradication of this disease has proven challenging. Although a number of drugs exist for the treatment of tuberculosis, 1.7 million people still die every year from this infection. The current treatment regimen involves lengthy combination therapy with four different drugs in an effort to combat the development of resistance. However, multidrug-resistant and extensively drug-resistant strains are emerging in all parts of the world. Therefore, new drugs effective in the treatment of tuberculosis are much-needed. The work presented in this thesis was focused on the early stages of drug discovery by applying different hit identification and hit expansion strategies in the exploration of two new potential drug targets, glutamine synthetase (GS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR). A literature survey was first carried out to identify new Mtb GS inhibitors from compounds known to inhibit GS in other species. Three compounds, structurally unrelated to the typical amino acid derivatives of previously known GS inhibitors, were then discovered by virtual screening and found to be Mtb GS inhibitors, exhibiting activities in the millimolar range. Imidazo[1,2-a]pyridine analogues were also investigated as Mtb GS inhibitors. The chemical functionality, size requirements and position of the substituents in the imidazo[1,2-a]pyridine hit were investigated, and a chemical library was designed based on a focused hierarchical design of experiments approach. The X-ray structure of one of the inhibitors in complex with Mtb GS provided additional insight into the structure–activity relationships of this class of compounds. Finally, new α-arylated fosmidomycin analogues were synthesized as inhibitors of Mtb DXR, exhibiting IC50 values down to 0.8 µM. This work shows that a wide variety of aryl groups are tolerated by the enzyme. Cinnamaldehydes are important synthetic intermediates in the synthesis of fosmidomycin analogues. These were prepared by an oxidative Heck reaction from acrolein and various arylboronic acids. Electron-rich, electron-poor, heterocyclic and sterically hindered boronic acids could be employed, furnishing cinnamaldehydes in 43–92% yield.