Efavirenz, a drug that is still inaccessible to millions of people worldwide, is potent non nucleoside reverse transcriptase inhibitor (NNRTI), is one of the preferred agents used in combination therapy for first-line treatment of the human immunodeficiency virus (HIV). NNRTIs attach to and block an HIV enzyme called reverse transcriptase, by blocking reverse transcriptase; NNRTIs prevent HIV from multiplying and can reduce the amount of HIV in the body. Efavirenz can't cure HIV/AIDS, but taken in combination with other HIV medicines (called an HIV regimen) every day helps people with HIV live longer healthier lives. Efavirenz also reduces the risk of HIV transmission and can be used by children who are suffering from HIV/AIDS. All the above therapeutic uses of efavirenz prompted us to identify the novel and hopefully cost efficient synthetic methodology for the preparation of efavirenz. In this thesis a new synthetic method for asymmetric synthesis of efavirenz is described. This route started from commercially available starting materials and it is first established in traditional batch chemistry and further the parameters transferred to a semi continuous flow protocol for optimization.

No description available.

Urease of Helicobacter pylori is an important virulence factor implicated in the pathogenesis of many clinical conditions, such as chronic gastritis, peptic ulceration, and gastric cancer. Many urease inhibitors have been discovered, like phosphorodiamidates, hydroxamic acid derivatives, and imidazoles. Despite good activities at the enzyme level and excellent kinetic properties most of them have not been used as therapeutic agents in vivo because of their side effects, toxicity and instability. This has led to much attention to focus on exploring the novel urease inhibitory activities of natural products because of their low toxicity and good bioavailability. Honey, a natural product has been used in folk medicine due to its antitumor, antioxidant, antimicrobial and anti-inflammatory properties. The aims of this study were to isolate, characterise, purify urease produced by H. pylori and investigate the inhibitory effects of solvent honey extracts on its enzymatic activity. Urease was found to be both surface-associated and cytoplasmic. Maximum cytoplasmic urease activity was found to occur after 72 hr whereas maximum extracellular urease activities were found to occur after 96 hr. Characterization of the crude cytoplasmic urease revealed optimal activity at a pH of 7.5 and temperature of 40°C. The kinetic parameters Vmax and Km were 45.32 U ml-1 and 61.11 mM respectively.The honey extracts inhibited the activity of the crude urease in a concentration dependent manner. The Lineweaver-Burk plots indicated a non-competitive type of inhibition against H. pylori urease. The two honey extracts gave promising inhibitory activities against urease of H. pylori. Thus the results of this study delineates that inhibition of urease can ease development in therapeutic and preventative approaches based on the enzymatic activity of this Helicobacter protein.

An enzyme activity located in the soluble fraction of rat liver which is known to deacetylate N8-acetylspermidine is studied. Using a crude cytosol preparation from rat liver, tritium-labelled N8-acetylspermidine [acetyl-3H] was shown to undergo deacetylation which was dependent upon both time of incubation and protein concentration. The Michalis constant for this deacetylation of N8-acetylspermidine is approximately 4.4 μM. N1-Acetylspermidine showed a competitive inhibition of this N8-acetylspermidine deacetylation activity, and was found to be the most potent inhibitor tested. Diamine and polyamine compounds were also shown to inhibit the deacetylation of N8-acetylspermidine. Spermidine was the most potent inhibitor of the naturally occurring polyamines tested, followed in order by spermine, putrescine, and cadaverine. Of numerous acetylated compounds studied, only acetylprocainamide showed any inhibition of this N8-acetylspermidine deacetylating activity at concentrations below 10.0 mM. The possible functions of the deacetylating activity are discussed along the speculatio of the role this enzyme activity may play in vivo.

Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH-dependent FabG into 3-hydroxyacyl-ACP, which is dehydrated by FabA to form enoyl-ACP. The NADH-dependent FabI (InhA) completes the cycle. Subsequent rounds of elongations in the pathways are catalyzed by the condensing enzyme FabF. For undecylprodiginine biosynthesis in S. coelicolor, homologues of the condensing enzymes (FabH and FabF) and the ACP (FabC) are encoded by redP, redR and redQ respectively in the red gene cluster. The genes encoding 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabA), and enoyl-ACP reductase (FabI), are putatively shared between fatty acid and undecylprodigine biosynthesis, since the corresponding genes are not present within the red gene cluster of S. coelicolor. RedP is proposed to initiate biosynthesis of undecylprodiginine alkane chain by condensing an acetyl-CoA with a malonyl-RedQ, in contrast to FabH which process a broad range of acyl-CoA with malonyl-FabC. The 3-keto group of the resulting 3-ketoacyl-RedQ is then reduced to provide butyryl-RedQ, presumably by the type II FAS enzymes FabG, FabA and FabI. These enzymes would not differentiate between straight and branched-chain substrates, and have equal preference for FabC and RedQ ACPs. RedR would then catalyze four subsequent elongation steps with malonyl-RedQ, with appropriate 3-keto group processing after each step. The proposed role and substrate specificities of condensing enzymes RedP and FabH have not been investigated in S. coelicolor. The genes encoding FabG, FabA, and FabI have not been characterized in Streptomyces. Analysis of the S. coelicolor genome sequence has revealed the presence of one fabI gene (SCO1814, encoding an enoyl-ACP reductase), and three likely fabG genes (SCO1815, SCO1345, and SCO1346, encoding β-ketoacyl-ACP reductase). In the current study the substrates specificities of both RedP and FabH were determined from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC) (FabC is a dedicated ACP for fatty acid biosynthesis and RedQ for undecylprodiginine biosynthesis in S. coelicolor). For RedP, activity was only observed with a pairing of acetyl-CoA and malonyl-RedQ. No activity was observed with isobutyryl-CoA consistent with the proposed role for RedP and the observation that acetyl CoA-derived prodiginines predominate in S. coelicolor. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonyl-FabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyl-FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyryl-CoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines. The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-ketoacyl-RedQ substrates for undecylprodiginine biosynthesis. Similar to FabG, spectrophotometric and LC/MS assays were also carried out to elucidate the functional role and substrate specificity of S. coelicolor FabI. The kinetic analyses demonstrate that SCO1814 has NADH-dependent enoyl-ACP reductase activity. Spectrophotometric and LC/MS assays demonstrated that FabI does not differentiate between straight and branched-chain substrates, and has equal preference for FabC and RedQ ACPs. These observations provide experimental support for the hypothesis that these enzymes are shared and process the intermediates in the elongation cycle of both fatty acid and undecylprodiginine biosynthesis. In summary, these studies have demonstrated the activity of enzymes RedP, FabH, FabG and FabI (InhA) previously uncharacterized in S. coelicolor and clarified their role in fatty acid and undecylprodiginine biosynthesis.

Structural properties of phosphoglucose isomerases isolated from a variety of species have been compared by peptide fingerprinting, predicted amino acid sequence homologies and by denaturation and renaturation studies. The enzymes are more readily denatured in guanidinium chloride than in urea, and the isomerase isolated from yeast is more stable toward acid pH than the rabbit muscle enzyme. The rates of guanidinium chloride-induced denaturation are markedly increased by ionic strength and decreased by substrates, competitive inhibitors or glycerol. The enzyme can be renatured, but only in the presence of glycerol. The renaturation process is dependent on protein concentration and temperature and provides a method for the formation of mixed species heterodimers.

No description available.

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Les analogues de nucléoside représentent une famille d'agents thérapeutiques très largement utilisée en chimiothérapie anticancéreuse. Cependant des phénomènes de résistance d'origine multifactorielle apparaissent chez les patients atteints de leucémie et il semble que la surexpression d'une enzyme, la 5'-nucléotidase cytosolique de type II (cN-II) soit un des facteurs impliqués. Nous nous sommes donc intéressés à la synthèse et à l'étude d'inhibiteurs potentiels de cN-II du type analogues de nucléoside phosphonate. Ce manuscrit rapporte dans un premier temps le contexte biologique des travaux de thèse, et les découvertes ayant supposés l'implication de la cN-II dans les mécanismes de résistance associés à l'utilisation clinique de nucléosides cytotoxiques. Le deuxième chapitre décrit la synthèse d'analogues phosphonates de l'uridine et de la cytosine modifiés en position 5, ainsi que leur évaluation biologique vis-à-vis de la cN-II purifiée. Enfin, la mise au point d'une stratégie inédite, via l'ouverture d'un époxynucléoside par un phosphite, a permis de synthétiser des béta-hydroxyphosphonates en série purique, et également d'obtenir les prodrogues bis(SATE) correspondantes. / Nucleosidic analogs are widely used as therapeutic agents in antitumoral chemotherapy. However, cellular resistance appears in a multifactorial manner in leukemic patient and it seems that the overexpression of a nucleotidase, the 5'-cytosolic nucleotidase (cN-II) is involved in this phenomenon. We focused our interest on the synthesis and the study of potential inhibitors belonging to the family of phosphonate nucleoside analogs. First, we reviewed literature data about the biological context of our research, especially concerning the involvement of cN-II in resistance phenomenon. Then, we described the synthesis of various phosphonate nucleosides bearing 5-modified uracil and cytosine as nucleobase, as well as their evaluation as inhibitors against the purified enzyme. Finally, a novel strategy for the synthesis of beta-hydroxyphosphonate nucleosides in purine series was designed and developed, and then applied to the synthesis of the corresponding bis(SATE) prodrugs.

A doença de Chagas afeta milhões de pessoas e os fármacos existentes não são seguros e apresentam eficácia limitada. Muitos produtos naturais mostraram efeitos inibitórios contra uma enzima importante para a sobrevivência do Trypanosoma cruzi, a gliceraldeído-3-fosfato desidrogenase (GAPDH). Dentre esses produtos naturais destacam-se aqueles da classe dos flavonoides, sendo que um deles, o tilirosídeo, mostrou-se interessante por inibir a enzima com valores de IC50 e Ki iguais a 46 e 25 µM, respectivamente, além de ter sido eficaz contra a cepa do T. cruzi resistente a fármacos (cepa Y) mostrando um valor de IC50 igual a 770 µM. Com objetivo de identificar novos potenciais inibidores da TcGAPDH baseados no tilirosídeo foram empregados métodos computacionais nos quais combinaram-se duas diferentes estratégias: os ensaios virtuais baseados na estrutura do ligante (LBVS) e os ensaios virtuais baseados na estrutura do receptor (SBVS). Os compostos que se ajustaram ao sítio catalítico da enzima e preditos para interagir de forma efetiva com o alvo foram adquiridos e testados contra a TcGAPDH. Os estudos de inibição enzimática foram realizados utilizando as técnicas de calorimetria de titulação isotérmica e espectroscopia de fluorescência obtendo como resultados as constantes de inibição dos compostos selecionados e seus modos de inibição. Dois flavonoides, o Nequimed 214 e o Nequimed 215 inibiram a TcGAPDH na mesma grandeza que o composto de partida, o tilirosídeo, mas como possuem cerca da metade da massa molecular, houve grande aumento da eficiência do ligante. A partir dessa informação, foram selecionados uma segunda geração de compostos preditos a interagir com a TcGAPDH. Deste modo, foram adquiridos bioisósteros de flavonoides que foram testados contra essa enzima, sendo que dois dos mesmos mostraram-se ativos e com alta eficiência do ligante. Foram adquiridos também compostos pertencentes à classe das hidantoínas, rodaninas, tio-hidantoínas e pirrolidina-2,4-dionas sendo que muitos dos mesmos foram ativos e mostraram os maiores valores de eficiência do ligante já relatados para a TcGAPDH, tornando-os excelentes candidatos para otimização molecular. Os resultados obtidos sugerem que vários inibidores possuem inibição não competitiva com relação ao substrato G3P. Os inibidores mais potentes foram testados contra a GAPDH de humanos e não foi verificada seletividade relevante. / Chagas disease affects millions of people worldwide. The available drugs are not safe and show limited efficacy. Many natural products inhibit the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important Trypanosoma cruzi enzyme. Among them, flavonoids have stood out and one of them, the tiliroside, inhibited the enzyme with IC50 and Ki values of 46 and 25 µM, respectively. Furthermore, unpublished results showed that this compound was effective against the drug-resistant strain of T. cruzi (Y strain) with IC50 value of 770 µM. In order to select potential inhibitors of TcGAPDH based on the tiliroside structure, computational methods were used combining two different strategies, the ligand-based virtual screening (LBVS) and the structure-based virtual screening (SBVS). The compounds predicted to interact effectively with the target and fit into the active site of the enzyme were purchased and tested. Enzyme inhibition studies were performed using isothermal titration calorimetry and fluorescence spectroscopy from which the constants and mode of inhibition of the compounds were determined. Two of the tested flavonoids, Nequimed 214 and Nequimed 215, showed inhibition activity against TcGAPDH in the same magnitude values as the starting compound, the tiliroside, in spite of their lower molecular weight, thus greatly enhancing the ligand efficiency (LE). These data prompted us to search for some flavonoid bioisosters that were obtained and tested against this enzyme, and two of them proved to be active with high ligand efficiencies. We also purchased compounds belonging to the class of hydantoins, pyrrolidine-2,4-dione, thio-hydantoin and rhodanine. Many of them were active at low micromolar concentration range and exhibited the highest ligand efficiencies ever reported for this enzyme, therefore becoming excellent candidates for molecular optimization. The obtained results suggest that most of inhibitors tested behave as non-competitive inhibitors with respect to the G3P substrate. The most potent inhibitors were tested against human GAPDH and relevant selectivity was not observed.