Dissertations / Theses on the topic 'Biochemistry|Pharmaceutical sciences'

Phenylketonuria (PKU) is an inborn error of metabolism characterized by a loss of phenylalanine hydroxylase activity; an enzyme that metabolizes phenylalanine to tyrosine. Phenylalanine ammonia lyase (PAL) is currently being evaluated as a possible therapy for the management of PKU. PAL catalyzes the conversion of phenylalanine to transcinnamic acid (TCA). Our proposed therapy involves encapsulation of PAL enzyme in ethyl cellulose microcapsules using the spray drying method. PAL activity is markedly reduced due to product inhibition. We hypothesized that the addition of albumin to the PAL reaction mixture would bind the TCA and prevent it from inhibiting PAL. In the first phase of our research, we developed an HPLC assay to quantitate phenylalanine and TCA in the presence of albumin. In the second phase, we determined that albumin completely alleviated product inhibition and enhanced PAL activity. Subsequent ultrafiltration studies showed that albumin acted by extensively binding to and sequestering TCA. PAL microcapsules will be taken orally. In the final phase, we studied the activity of encapsulated PAL in simulated GIT conditions to evaluate the ability of microcapsules to protect PAL enzyme against pH and protease mediated degradation. The activity of encapsulated PAL was lower than an equivalent amount of free PAL possibly due to diffusional limitations to the entry of phenylalanine into the microcapsules. Encapsulation of PAL in ethyl cellulose microcapsules did not protect against acidic pH mediated reduction of PAL activity or pepsin mediated proteolytic degradation.

Human epidermal growth factor receptor 2 (HER2) is a well-studied therapeutic target as well as a biomarker of breast cancer. HER2-targeting affibody (Z _HER2:342) is a novel small scaffold protein with an extreme high affinity against HER2 screened by phage display. However, the small molecular weight of Z_HER2:342 has limited its pharmaceutical application. Human serum albumin (HSA), as the main protein in plasma, has been commonly used to extend the small peptides serum half-life. Its high solubility, stability and excellent ability to carry multiple ligands in blood stream make it a good candidate for drug delivery.

Two HSA and Z_HER2:342 fusion proteins, one with a single Z _HER2:342 domain fused to the C terminus of HSA (rHSA-ZHER2) and the other with two tandem copies of Z_HER2:342 (rHSA-(ZHER2)₂), have been constructed, expressed, and purified. Both fusion proteins possessed the HER2 and fatty acid (FA) binding abilities demonstrated by in vitro assays. Interestingly, rHSA-(ZHER2)₂, not rHSA-ZHER2, was able to inhibit the proliferation of SK-BR-3 cells at a relatively low concentration, and the increase of HER2 and ERK1/2 phosphorylation followed by rHSA-(ZHER2) ₂ treatment has been observed. However, the inhibition effect on HER2-overexpressing cells is cell linedependent. Fusion protein rHSA-(ZHER2)₂ showed preferred accumulation in tumor tissues in xenograft model.

HSA fusion proteins are easy and economical to express, purify, and formulate. Two formulation strategies have been explored, one is to complex the fusion protein with FA modified chemo drugs, and the other is to make them into nanoparticles. As expected, HSA fusion proteins and fusion protein-bound fatty acid-modified fluorescein isothiocyanate (FITC) could be efficiently taken up by cells. FA-Taxol/albumin formulation showed its advantages over Taxol/albumin treatments on in vitro cell growth inhibition. Nanoparticles containing rHSA-ZHER2 produced by desolvation method displayed optimal size distribution, satisfactory stability and preferred binding/uptake on HER2-overexpressing cells. These results proved the feasibility of using HSA fusion proteins as therapeutic agents as well as carriers for targeted drug delivery.

Inappropriate expression of nicotinic acetylcholine receptors in the central nervous system is associated with nicotine addiction, Alzheimer’s disease, Parkinson’s disease and other disorders. Modulators (drugs) have the potential to restore circuit properties that arise from inappropriate expression of nicotinic receptor’s. Compounds that interact with allosteric sites have a distinct advantage over agonists and partial agonists, in that, they retain normal activation patterns by allowing binding of the endogenous ligand. Positive allosteric modulators boost the receptors ability to respond to agonist. Studies of these modulators constitute a first step toward the identification and development of better compounds that minimize signaling errors at cholinergic synapses. We have used single molecule methods to investigate the action of a novel positive allosteric modulator, desformylflustrabromine (dFBr), on nicotinic receptors. Our studies were focused on the α4β2 subtype of nicotinic receptors in the brain. These receptors exist in two forms with low sensitivity (α4₂β2₃) or, alternatively, high sensitivity (α4₂β2₃) to agonist. Our experiments allowed us to develop detailed gating models for high and low sensitivity receptors, as well as gain new insights regarding the mechanisms that underlie potentiation by allosteric modulators. We found that dFBr potentiates low sensitivity receptors by destabilizing desensitized states of the receptor. In contrast, potentiation of high sensitivity receptors arises from a synchronization of openings following an application of agonist due to an increase in the opening rate. Based on our results we now have a better understanding of the advantages of dFBr on high and low sensitivity receptors.

The aim of this thesis is to evaluate the impact of somatostatin receptor subtype-4 (SSTR4) actions on microglia cell viability, towards the understanding and advance of pharmacological treatments for Alzheimer’s disease (AD). As of March 2016, there were 5 million people living in the United States with AD. Current drugs for AD have highly variable effects from patient to patient and are palliative at best. This thesis project focuses on the study of the BV2 cell line and the compound NNC 26-9100 (NNC). BV2 cells are immortalized microglial cells that maintain most of their morphology. The data collected suggests that BV2 cells can phagocytize amyloid-? peptides (A?), respond to lipopolysaccharide (LPS), and have the somatostatin receptor subtype-4 (SSTR4). NNC is a selective agonist of the SSTR4 and we have found that it causes BV2 cells to increase in number. The effects of NNC were able to be reduced with a somatostatin receptor pan-antagonist, cyclosomatostatin, and the adenyl cyclase activator forskolin. NNC can alter BV2 numbers by binding to the SSTR4, creating an intracellular cascade that results in the inhibition of adenyl cyclase and an increase in cell count. Collectively, a potential therapeutic mechanism for AD is increasing the number of microglial cells to increase both amyloid beta (A?) phagocytosis and degradation of A? by neprilysin.

Recent strategies for anticancer drug design have been focused on utilizing antibody as a drug or targeted moiety for targeted drug delivery. Antibody−drug conjugates (ADCs) have become a promising new class of targeted therapeutic agents for treatment of cancer. ADCs are designed to preferentially direct a cytotoxic drug to a cell-surface antigen recognized by an antibody. However, there are some challenges in developing ADCs, such as limited solid tumor penetration, high manufacturing costs and antibody-drug stoichiometry. Smaller molecules such as peptides have been shown to specifically bind to cancer related targets. These peptides can be used to form peptide-drug conjugates (PDCs) to overcome above-mentioned drawbacks presented by ADCs. In this study, it was hypothesized that novel synthesized PDCs can be a strategy for breast cancer therapy. HER2 specific binding peptides, MARAKE and MARSGL, were modified by addition of a cysteine at C-terminus. The modified peptides were coupled with monomethylauristatin E (MMAE) by using maleimidocaproyl (MC) as a non-cleavable linker to form peptide-drug conjugates (YW1, YW2) and maleimidocaproyl-valine-citrulline (MC-VC) as a cleavable linker to form peptide-drug conjugates (YW3 and YW4). The peptides, peptide-drug conjugates and MC-MMAE, MC-VC-MMAE were characterized using ESI-MS and purified by using high-performance liquid chromatography (HPLC). Cellular uptake study was performed to determine binding specificity and internalization of two HER2 specific peptides and cysteine-modified peptides (MARAKEC, MARSGLC). In vitro cell viability assay was conducted to assess the cytotoxicity and determine the targeting specificity as well as the potency of the peptide-drug conjugates. The purity of each compound was greater than 90%. Internalization of both HER2 specific binding peptides and cysteine-modified peptides were significantly higher than random peptides in HER2 over-expressed cell lines, MDA-MB361 and ZR75, while negligible uptake in HER2 negative cell line, HEK293. MC linked PDCs showed similar cytotoxicity as peptide in all cell lines; while MC-VC linked PDCs have higher cytotoxicity than MMAE in HER2 positive cell line and significant lower cytotoxicity than MMAE in normal cell line HEK293. However, PDCs with MC link do not show significant difference in cytotoxicity compared to the peptide in all cell lines. In conclusion, specificity of HER2 binding for both peptides was preserved after modification with cysteine. The derivation of MMAE to link drug and peptide played a crucial role in the anticancer activity. Peptide-MMAE conjugates with cleavable linker showed a promising targeting capability for delivery of MMAE to HER2 overexpressed cancer cells.

Gamma-Hydroxybutyrate (GHB) is an endogenous short-chain fatty acid formed from Gamma-aminobutyric acid (GABA). Clinically, GHB is marketed in the United States as Xyrem to treat narcolepsy with cataplexy and in Europe for the treatment of alcohol withdrawal and narcolepsy. However, the illicit use and abuse of GHB occurs due to its sedative/hypnotic and euphoric effects. Monocarboxylate transporters (MCTs and SMCTs) are integral membrane proteins that control the bidirectional transport of endogenous substrates including lactate, acetate and pyruvate. They have also been found to transport and mediate the clearance and distribution of GHB. MCTs demonstrate a wide tissue distribution, including brain, kidney, liver, and intestine, all of which play an important role in determining the disposition of GHB. Sex differences in drug elimination pathways contribute to the wide range of inter-individual variability observed between sexes with respect to drug disposition and effect. Sex differences in MCT expression have been observed in the brain, muscle, liver and kidney with variations potentially driven by sex hormones; however, there is an absence of information on how these expression differences translate into sex differences in GHB toxicokinetics. The objective of this study was to evaluate sex differences and the influence of the estrus cycle on GHB toxicokinetics after IV administration. Our hypothesis is that renal clearance and toxicokinetics will vary over the estrus cycle. Estrus cycle stage in female rats was determined by vaginal lavage prior to GHB administration. Ovariectomized (OVX) females were included in the study to evaluate GHB toxicokinetics in the absence of female sex hormones. Our results demonstrated that sex and the estrus cycle influence GHB toxicokinetics. Total and renal clearance varies over the estrus cycle with the highest renal clearance observed in proestrus females. In contrast, males and OVX females demonstrated significantly lower renal clearance. These results suggest that GHB toxicity and risk of overdose varies over the estrus cycle due to expression changes in renal MCTs and SMCTs. Future studies will evaluate higher GHB doses to determine the role of sex hormones in GHB overdose and fatality. In addition, hormone replacement studies will be conducted to confirm the role of individual sex hormones on GHB toxicokinetics.

The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants were designed to selectively increase and decrease stability by relating KS propensities with changes to α-helical structure. This study suggests that the sockets from the KS Model can be used as a measure of α-helical structure and stability. The KS model was also used to investigate coiled-coil specificity in bZIP proteins. Identifying and characterizing the interactions that determine the dimerization specificity between bZIP proteins is a crucial factor in better understanding disease formation and proliferation, as well as developing drugs or therapeutics to combat these diseases. Knob-Socket mapping methods identified Asn residues at a positions within the helices, and were determined to be crucial factors in coiled-coil specificity. Site-directed mutagenesis was conducted to investigate the role of the Asn residues, as well as the role played by the neighboring residues at the g and b positions. The results indicate that the Asn at the a position defines coiled-coil specificity, and that the Knob-Socket model can be used to determine bZIP protein quaternary interactions.

Hepatitis C virus (HCV) is the etiologic agent responsible for the majority of cases of non-A, non-B hepatitis. It is estimated that 2% of the world's population is infected with hepatitis C with roughly 75% of infections progressing into a chronic state. Current interferon therapy is largely ineffective against chronic Hepatitis C due to the induction of interferon-resistance by HCV. The underlying mechanisms for resistance to interferon therapy have been widely studied and documented and as is the case in interferon resistant malignancies, there is some evidence that HCV infected cells are more susceptible to infection by interferon sensitive viruses. Most current research into novel HCV therapeutics has focused on the discovery of inhibitors of viral replication. However, the high mutation rate of the HCV genome makes it a virtual certainty that viral variants resistant to protease and/or polymerase inhibitors will arise. As an alternative approach, we have designed a therapeutic strategy that exploits both the expression of a viral protease in infected cells as well as the defects in the interferon pathway to build selectively active therapeutics that target and kill HCV infected cells. Initial experiments showed some promise using this approach and indicated areas for further development. Conditionally replicating viruses failed to rescue in HCV expressing cell lines, thus requiring further modification. A separate approach involving conditional stabilization of the suicide gene HSV-1 TK was successful with a two-fold increase in the expression levels of TK in cells co-expressing HCV protease.

Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE). Anticoagulants are very important for the treatment of thrombotic diseases. The adverse effects associated with current clinically used anticoagulants warrant the continuous search for new agents. Thrombin, being the central player in the coagulation cascade, remains a very important target for anticoagulant therapy, however drugs inhibiting its activity carry the risk of prolonged bleeding. Based on a previously identified sulfated benzofuran thrombin inhibitor, we have developed analogs with submaximal inhibition of the protease. These agents inhibit thrombin with efficacies approaching 50%, for both chromogenic and macromolecular substrates, ensuring a basal level of thrombin activity even at saturating inhibitor concentrations. The most potent of these compounds had a potency of 1.8 µM, 2-3 fold better than the lead. Additionally, these compounds utilize an allosteric mechanism for thrombin inhibition. Further, studies have revealed structural features responsible for submaximal thrombin inhibition. Fibrinolysis is an important part of hemostasis and plasmin is the most important fibrinolytic enzyme. Anti-plasmin agents are thus important for conditions such as hemophilia; however, there are no clinically used direct plasmin inhibitors. By structural modifications of a previously identified sulfated diflavonoid plasmin inhibitor, we have achieved a compound with 12-fold better potency (IC50 = 6.3 ± 0.4 µM), and a selectivity index of at least 22 over closely related serine proteases. We have shown that this compound inhibits plasmin mediated clot lysis, and further demonstrated that its activity is reversible using protamine sulfate, indicating its potential as a lead for the development of clinical anti-plasmin agents. HNE, a serine protease associated with inflammatory diseases is known to be inhibited by GAGs. However, the interactions at the molecular level have remained elusive. Using biochemical methods, and by studying the inhibitory potency of different GAGs and GAG oligosaccharides, we have shown that an octasaccharide may be the ideal GAG length for the achievement of potent HNE inhibition. Under our assay conditions, the inhibition of HNE by an octasaccharide species was only 5-fold less than that of unfractionated heparin, whereas the hexasaccharide species was 30-fold less active. The data also suggests that the inhibition of HNE by GAGs is via an allosteric mechanism and using molecular modeling, we have identified putative GAG binding sites on HNE and further identified GAG species with potential selectivity for anti-HNE activity

Aryl hydrocarbon receptor (AHR), commonly known as an environmental sensor involved in the metabolism and elimination of xenobiotic substances, is also an important modulator in the development and functioning of the immune system. AHR expression is varied in the T cell subsets with the highest expression in T-helper 17 and T regulatory cells. Work from many researchers has suggested that AHR can act as a tumor promoter or a tumor suppressor depending on the tumor type. Our goal is to understand the role played by AHR in MC38 syngeneic colon carcinoma tumor model. In the absence of AHR, MC38 tumor progresses by an increase in tumor associated macrophages (TAMs), M2 macrophages and a decrease in CD8a positive cytotoxic lymphocytes. Analysis of the intratumoral cytokines reveals a pro-inflammatory phenotype. This has been assessed by pharmacologic blocking of the receptor using CH223191 and in AHR deficient (AHR-/-) mice. Therefore AHR acts as a tumor suppressor gene in colon carcinoma tumor model and silencing it may lead to colon cancer progression.

In the past, aminocyclohexanol rings have been successfully utilized as pH-triggered molecular switches in various trans-2-aminocyclohexanol derivatives. By changing the groups on the amine nitrogen, these models provided a wide pH range in which a switch can occur. The pH-induced switch of conformation was monitored by 1H-NMR spectroscopy. The models were also incorporated into the bilayer membrane of liposome structures and tested for their ability to disrupt their membrane upon their conformational flip induced by a decrease in pH. In this work, the amide bond has been studied as a molecular switch and various amide derivatives have been tested for their potential as lipid-like compounds that also exhibit a pH-sensitive conformational flip. The conformational analysis of these compounds was achieved by various NMR techniques and NMR acid-base titration studies were utilized to estimate the pKa of a number of the compounds described.

L'imagerie par spectrométrie de masse MALDI est une technologie actuellement en plein essor. Elle permet d'obtenir rapidement en une seule étape d'acquisition la répartition moléculaire de différents composés (en particulier protéines) présents dans un tissu ; en s'affranchissant des étapes longues et coûteuses en échantillons que sont les extractions et séparations habituellement nécessaires par des techniques plus classiques.
Cependant, afin d'augmenter encore la potentialité de cette technologie, des développements restent encore à effectuer. Les recherches menées ont donc plus particulièrement portées sur ces développements.
En particulier, la recherche et l'étude de nouvelles matrices plus adaptées à l'analyse directe de tissu en MALDI sont particulièrement importantes. Dans ce contexte, certaines matrices ioniques se sont révélées particulièrement adaptées aux tissus en permettant d'obtenir une plus grande intensité du signal, un plus grand nombre de composés détectés, de bonnes performances en mode négatif, une grande homogénéité de cristallisation, une grande stabilité sous vide et une faible ablation de matériel consécutivement à l'irradiation laser. Dans un autre aspect, le traitement préalable des tissus permet également une amélioration de la qualité spectrale et des performances d'études structurales en mode MS/MS. Se sont révélés particulièrement intéressants les traitements des tissus aux solvants organiques et les digestions enzymatiques et en particulier pour les tissus conservés en blocs de paraffine après fixation.
D'autre part l'étude de la répartition des ARNm au sein des tissus est un développement crucial afin d'obtenir des images de colocalisation transcriptome/protéome. Est proposé dans ce travail un nouveau concept permettant de réaliser ces images, basé sur une analyse indirecte des ARNm, au travers de l'utilisation d'un groupement photoclivable relié à un peptide marqueur de séquence connue qui sera détecté.
Enfin, l'ensemble de ces développements trouve de nombreuses applications dans le domaine de la biologie et notamment dans le cadre de pathologies tel que le cancer de l'ovaire.

Polyamines are ubiquitously present in all living cells. The abnormal metabolism of polyamines that is associated with certain types of cancers has been the focus of several investigations. Enzymes that are involved in the transacetylation of polyamines have been studied extensively, so as to develop inhibitors of these enzymes which may be used as drugs in cancer therapy. Based on indirect evidence, the nuclear spermidine acetyltransferase has been thought to be a critical enzyme that is associated with genetic derepression leading to cancerous growth. In the present study a novel, rapid, sensitive and highly reproducible radio chemical procedure has been developed for assaying spermidine (polyamine) acetylation. The study contains data showing range of linearity of the procedure, percent product recovery, as well as low interference from the unreacted acetyl coenzyme A. Rat liver nuclear spermidine acetyltransferase has been purified using the biochemical procedures annmonium sulfate precipitation, DEAE chromatography, Hydroxyapatite chromatography, Diaminobutyl agarose chromatography and Polyacrylamide P-300 gel filtration. The enzyme obtained at the end of such procedures was found to be essentially homogeneous as seen on native gel electrophoresis. The purified enzyme has been shown to have an isoelectric point of 5.2. Bicine and Hepes were found to be more suitable as buffering species for good enzyme activity. The enzymatic reaction velocity was found to increase with temperature upto 36$\sp\circ$C and was found to increase linearly up to four minutes under non limiting conditions in the presence of 20% glycerol. Using the purified enzyme it has also been established that of the three nuclear polyamines, spermidine is the preferred substrate. The apparent Km for acetyl Co A with spermidine as substrate was found to be about 5 mM. The purified enzyme does acetylate histones. All the substrate analogs containing aminobutylamino group are acetylated by the enzyme.

Chemistry
Ph.D.
The synthesis of pyridinium cationic lipids, their counter-ion exchange, and the transfection of lipoplexes consisting of these lipids with firefly luciferase plasmid DNA (6.7 KDa), into lung, prostate and breast cancer cell lines was investigated. The transfection ability of these newly synthesized compounds was found to be twice as high as DOTAP/cholesterol and LipofectamineTM (two commercially available successful transfection agents). The compaction of the DNA onto silica (SiO2) nanoparticles was also investigated. For this purpose, it was necessary to study the stability and fusion studies of colloidal systems composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), a zwitterionic lipid, and mixtures of DMPC with cationic DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane).
Temple University--Theses

The aryl hydrocarbon receptor (AHR) is a transcription factor first discovered to be activated by exogenous ligands, such as dioxins, and helps promote downstream gene (e.g. CYP1A1) transcription to metabolize the toxicants. With the reports of various AHR targets genes, the expression levels and activities of AHR have been implicated in many physiological and pathological situations. Understanding how AHR protein level is regulated would provide more information to target AHR. AHR stays in the cytosol in the absence of ligand in a complex with HSP90, p23 and XAP2. After ligand activation, AHR translocates into the nucleus, fulfilling its transactivation function and then is finally degraded by proteasomes. Here, we discovered a new mechanism that controls basal AHR protein level: the selective autophagy. Loss of AHR co-chaperone p23 leads to increased protein degradation of AHR through autophagy in HeLa cells. Inhibition of autophagy using several inhibitors (chloroquine, bafilomycin A1 or 3-methyladenine) increased AHR protein levels. Knocking down of key macroautophagy protein LC3B increases AHR protein levels and decreases the responsiveness of AHR to CQ treatment. The interaction between AHR and LC3B as well as AHR and autophagy receptor p62 were confirmed in vitro and in situ. AHR is found to be lysine (K) 63-ubiquitinated in HeLa cells, which is a common signal for the autophagy-lysosomal degradation.6 We also discovered that AHR is controlled by glycogen synthase kinase 3β (GSK3β) phosphorylation. Inhibition of GSK3β activity or its expression level increased AHR protein levels while expression of HA tagged-GSK3β lowers AHR protein levels. AHR protein level is regulated through autophagy. We confirmed the GSK3β-mediated phosphorylation of AHR by phos-tag gel electrophoresis couples with Western blot analysis and identified three putative phosphorylation sites of AHR in the C-terminal half of AHR sequence. Moreover, phosphorylated AHR constitutes the active pool for transactivation and phosphorylation tagged AHR for the autophagy-lysosomal degradation, which may act as way to limit its function.

The thesis describes utilizing mass spectrometry and computational methods to study two groups of molecular systems: small organic molecules and oligopeptides. The gas-phase acidities were measured and the structures of the molecular species were calculated. The small molecules investigated included methylparaben, ibuprofen, and triclosan, all known to have some biological activity. The gas-phase acidity measurements made for these small molecules had the solvent and collisional gas pressures adjusted in order to observe their potential influences. The results obtained provide insight into the ion chemistry of these molecules and how the energetics may change the observed behavior of the ion as well as the resulting thermochemical properties measured. The oligopeptides studied were a family of tri-peptides in which a cysteine probe was placed within an alanine backbone. The cysteine probe was either in the L- or D- configuration in order to detect any fundamental differences among the diastereotopic peptides. Compared to the L-cysteine isomers, the D-cysteine peptides appear to display a change in gas-phase behavior and their respective dissociation profiles. These changes may have an implication of altering the biochemical properties when chirality changes in biological systems.

In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of polyUb chain a substrate gets labeled with can determine the subsequent activity of that substrate. Substrate ubiquitination is achieved through an enzymatic cascade. First, an E1-activating enzyme activates a free Ub moiety. Then Ub is transferred onto an E2-conjugating enzyme, and finally an E3 ligase interacts with both substrate and E2~Ub complex to facilitate Ub transfer onto a substrate. Within this scheme, the E2-enzyme acts as a master manipulator in that, it controls when, where and how a ubiquitin chain is transferred onto a substrate. Irregular activity of E2-conjugating enzyme has been implicated in a wide variety of diseases such as cancer, neurodegenerative diseases, muscular dystrophy, genetic azoospermia and more. While attempts have been made to inhibit other ubiquitination cascade enzymes such as E3 ligases and E1-activating enzymes, there is a strikingly small number of inhibitors specifically targeting E2 enzymes mainly due to the high degree of structural conservation that exists among members of the E2 enzyme family. In this work, we introduce 3 novel linked-domain protein inhibitors of the E2-conjugating enzyme Ube2D. We covalently attached either UHRF1 RING domain or an affinity optimized U-box domain, with UHRF1 UBL domain or UbvD1.1 (A ubiquitin variant specific for Ube2D), through a glycine-serine linker, producing 3 unique inhibitors: Ring-UBL (RU), U-box-UBL (UU), and U-box-UbvD1.1 (UUD1.1). In this way, we attempt to specifically inhibit Ube2D for two purposes : 1) While Ube2D can interact with the largest number of E3 ligases and facilitate the largest number of polyUb chains, very little is known about cellular phenotypes specifically associated with Ube2D; 2) We want to establish whether targeting the E2 enzyme in general can be utilized as a viable therapeutic treatment for cancer. We show that all three inhibitors are able to inhibit ubiquitin assays using Ube2D and using ITC we measured binding affinities of UUD1.1 (5 nM) > UUWT (300 nM) > RU WT (3 µM). Furthermore, we found that all inhibitors could prevent E1, E3 and backside binding domain interactions simultaneously, which single domain UBL could not. UU and RU showed specificity towards Ube2D when tested against APC/C and Cullin1 E3 ligases and their cognate E2 enzymes. We propose that linking domains in this way, by targeting the backside binding domains of E2 enzymes, could be a strategy that can be standardized and applied to the rest of the E2 enzyme family as well. In vivo testing must now elucidate whether these inhibitors can provide more information about the cellular role of Ube2D and whether it is a viable therapeutic target to treat cancer.

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Immunoglobulin (Ig) light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African Americans, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 years causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the characterization of AG10 , a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10 , combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. The second part of the thesis discusses harnessing TTR as a platform to enhance in vivo half-life of therapeutic peptides. The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of a model peptide Gonadotropin Releasing Hormone (GnRH) and its analog GnRH-A without compromising their potency. Apart from GnRH, we have used other peptides to study their proteolytic stability in vitro . Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo . We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

Exposure of DNA to reactive oxygen species (ROS) results in the modified nucleobases (lesions) as well as strand scissions under physiological conditions. Due to its lowest oxidation potential (1.29 eV), guanine is the most easily oxidisable nucleobase. Furthermore, it has been observed that the 5'-guanine in G-tracts (e.g. GGG) has even lower oxidation potential (1.00 V vs. NHE). One of the representative G-rich examples is telomeres that consist of repeating units of 5'-d [TTAGGG]-3' found at the ends of chromosomes. Telomeres play an important role in biological functions, serving as guardians of genome stability; however, their G-rich nature implies that they can be readily oxidized. So how does nature protect these biologically important regions from oxidation? We believe the formation of a secondary structure known as G-Quadruplex in telomeric regions can partly serve as a protective role. In the first part of this work, we investigated DNA G-Quadruplex damage under various oxidation conditions and compare the damage results with single-stranded telomeric sequences. Damage to G-Quadruplex is generally less than single strands and is condition dependent. Guanines are the primary damage sites, but damage of adenine and thymine is also possible. Based on our studies, telomeric DNA can be readily oxidized to produce DNA lesions. How do DNA lesions affect the conformation and the stability of telomeric G-Quadruplex DNA? In the second part, we sought to address this question using various biophysical methods. Several native (OxodG, OxodA, and abasic site) and non-native (8-NH 2 -dA and 8-Br-dA) lesions were tested. UV thermal denaturation and circular dichroism revealed that the conformation and the stability of G-Quadruplex DNA are dependent on the location and the type of lesion in the sequence. G-Quadruplex DNA containing OxodG maintains its conformation with a decreased stability. Abasic site in the TTA loop affects the conformation of G-Quadruplex DNA but shows little effect on its stability. An unexpected stabilization of telomeric G-Quadruplex DNA was observed when deoxyadenosine (dA) in the loops was replaced with its native oxidized form OxodA. This is the first example of native DNA lesion that increases the stability of G-Quadruplex DNA. Like OxodA lesion, 8-NH 2 -dA (a non native DNA lesion) increases the stability of G-Quadruplex DNA while 8-Br-dA only affects the stability in KCl but has no significant effect in NaCl. In addition, studies of the effect of OxodA lesion on the human telomerase activity using TRAP assay will be discussed.

The low redox potential of guanines (G 1.29 V vs. NHE) compared to other nucleobases, makes them potentially susceptible to attack by exogenous and endogenous damaging species. This property of guanine has also been utilized for the development of several anticancer agents including the well-known platinum complexes, cisplatin and carboplatin. The two closely related nickel complexes, NiCR and NiCR-2H, exhibit significant differences in cytotoxicity towards MCF-7 cancer cells. In the first part of this work, we explain this difference using biochemical and biophysical approaches to study their interactions with duplex DNA. The nickel complexes were found to selectively oxidize guanines in bulged DNA structures in the presence of oxidant and notably NiCR-2H oxidizes guanines more efficiently than NiCR. According to 1 H NMR studies, NiCR-2H binds strongly to the N7 position of dGMP compared to NiCR and could be an important oxidation product of NiCR under physiological conditions. The second part of this work focuses on the secondary DNA structures known as G-quadruplex formed in the guanine rich telomeric region. G-quadruplex is formed by stacking of G-quartets (a coplanar cyclic array of four Gs) on top of each other. Its formation is known to inhibit the activity of the reverse transcriptase telomerase that is overexpressed in 80-90% cancer cells. The guanines in telomeric DNA are readily oxidized due to their low redox potential and the major oxidation product is 7, 8-dihydro-8-oxoguanine (OxodG). OxodG (0.58 V vs. NHE) can further be oxidized in the presence of one electron oxidants and the resulting product forms adducts with endogenous nucleophiles such as spermine. In light of these findings, we hereby designed and synthesized novel bifunctional perylene derivatives that can selectively bind to the telomeric DNA via G-quadruplex formation and subsequently react with OxodG in close proximity. These compounds have strong binding affinity towards G-quadruplex and can significantly stabilize the OxodG containing G-quadruplex motif by end stacking on the upper G-quartet. The effect of these compounds on telomerase activity and cytotoxicity towards Hep3B cancer cells was also evaluated.

Post-translational modifications (PTMs) of proteins play significant roles in regulation of biological activities and signal transduction. Examining their diversity is critical for understanding the mechanisms of cellular regulations. Among the various techniques employed for identification of PTMs, mass spectrometry has become a more and more important tool for detecting and mapping these covalent modifications and quantifying their changes. The two projects described in this dissertation focus mainly on the method development for characterization of two major PTMs, disulfide bonds and glycosylation. In the first project, the disulfide bond pattern of a rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha was assigned unambiguously based on a multi-enzyme digestion strategy in combination with MALDI-TOF mass spectrometry analysis. The disulfide bond pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. More importantly, an interesting phenomenon of gas-phase scrambling of disulfide bonds was observed during MALDI mass spectrometry analysis and a possible mechanism for this surprising scrambling was proposed. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. In the second project, the glycosylation of a glycoprotein ZPA from the vitelline envelope of Xenopus laevis was determined by applying a strategy of general proteolysis coupled with mass spectrometry. The vitelline envelope glycoproteins were first separated through SDS-PAGE. A nonspecific in-gel pronase digestion was performed on the excised band of ZPA to produce informative small glycopeptides. Lectin affinity chromatography was used for the enrichment of these glycopeptides. An in-gel PNGase F digestion was also carried out to release the N-linked glycans from ZPA. The enriched glycopeptides and glycans were finally analyzed by MS and MS/MS techniques on MALDI-TOF and MALDI-TOF/TOF instruments.

Cancer has been a pervasive and deadly problem for many years. No treatments have been developed that effectively destroy cancer cells while also keeping healthy cells safe. In this work, the knob-socket construct is used to analyze two systems involved in cancer pathways, the PDZ domain and the Bcl-BH3 complex. Application of the knob-socket model in mapping the packing surface topology (PST) allows a direct analysis of the residue groups important for peptide specificity and affinity in both of these systems. PDZ domains are regulatory proteins that bind the C-terminus of peptides involved in the signaling pathway of cancer progression. The domain includes five -strands, two -helices, and six coils/turns. In this study, the PST of all eight solved crystal structures of T-cell lymphoma invasion and metastasis 1 (Tiam1) PDZ domains are mapped to reveal details of ligand-domain binding pockets and packing interactions. Four main interactions were identified in the comparison of the PST maps and a consensus sequence was calculated using knob-socket interaction data. In the case of the Bcl-BH3 complex, binding of these two proteins prevents an unhealthy cell from undergoing apoptosis. In the knob-socket mapped protein-ligand interactions, the helical ligand consists of 8 to 10 residues that specifically interact with four helices on the binding protein: the N-terminus of Helix2, the main bodies of Helix3 and Helix4 and the C-terminus of Helix5. Among all of the interactions that were analyzed, there were three amino acids from the ligand, glycine, leucine, and isoleucine, that always packed into the hydrophobic groove that is key for ligand recognition. By using knob-socket analysis to map quaternary packing structure, it was possible to identify the quaternary-level protein interactions that define ligand specificity and binding strength. From this analysis, possible protein mimetics can be developed that could be used as cancer treatments.

Le souhait de réduire des effets secondaires associés aux anticancéreux a mené à considérer l'utilisation de prodrogues activables au site d'action, comme la cyclophosphamide (CPA). La CPA est activée majoritairement par le CYP2B6 humain avec une faible efficacité, obligeant à l'utilisation de concentrations importantes de prodrogue. Celles-ci peuvent être réduites par transfection au niveau de la tumeur d'un gène codant pour un P450 optimisé, possédant une efficacité catalytique élevée vis-à-vis de la CPA tout en étant le moins immunogène possible. Pour ce faire, en partant de la modélisation du CYP2B6 et du CYP2B11, forme canine à bas Km pour l'activation de la CPA, un gène synthétique du CYP2B11 pour l'expression dans la levure a été dessiné et divisé en 15 "modules" structuraux. Quinze chimères à points de jonction définis entre les deux CYP2Bs ont ensuite été construites par échange de ces modules, via une méthode originale de génération de banques ordonnées de chimères, SIGNAL, indépendante de l'homologie de séquence des enzymes parentaux. SIGNAL, à mi-chemin entre les processus classiques d'évolution dirigée et de mutagenèse dirigée, nous a permis, après analyse fonctionnelle des chimères, de mieux comprendre le mécanisme de métabolisation de la CPA par les deux enzymes et d'identifier des modules structuraux jouant potentiellement un rôle important dans la haute affinité du CYP2B11. En parallèle, la mise au point d'un système de sélection à haut débit des variants les plus efficaces pour l'activation de la CPA dans la levure, basé sur le principe du gène rapporteur, a été débutée, afin de pouvoir raffiner l'optimisation du P450 par un processus de mutagenèse aléatoire.

Over the past two decades, protein kinases have been intensively investigated as targets to treat neoplastic diseases. Many protein kinase inhibitors not only have therapeutic potential but are becoming invaluable reagents for the study of cell signaling. We aspired to use our Cyclin-Dependent Kinase 8 inhibitor, Q-12, as a probe for biomarker discovery for CDK8 inhibitor sensitive tumor types. Q-12 shows potent inhibition of cell viability and induction of apoptosis process in some triple-negative breast cancer and colorectal cancer cell lines in vitro. Western blot results indicate that the reduction of STAT1 phosphorylation could be a robust indicator of CDK8 target engagement in all three cancer cell lines used upon Q-12 treatment. Q-12 treatment of triple-negative breast cancer cell line (MDA-MB-468) decreases STAT1 phosphorylation but increases STAT3 phosphorylation. Q-12 activity in MDA-MB-468 cell is dependent on the activation of STAT3 phosphorylation. All results suggest that there may be a critical STAT1 to STAT3 ratio that may serve as a biomarker for CDK8 inhibitor sensitivity. In this precision medicine era, the discovery of biomarker is urgently needed to minimize the risks of severe side-effects by traditional chemotherapy and improve diagnosis and monitor therapy response across a wide spectrum of disease, especially heterogenous type of disease, like cancer.

The traditional anticancer drugs are distributed in vivo through systemic blood circulation with a very small portion reaching the tumor site. Targeted drug delivery systems are developed in efforts to concentrate the drug molecules in the tissue of interest while reducing the drug distribution to healthy tissues to reduce the side effects. Liposomes are colloidal systems composed of amphiphilic molecules that assemble into vesicle structures in aqueous media. They are common carriers for targeted drug delivery with the advantages of low toxicity, low immunogenicity and the ability of encapsulating both lipophilic and hydrophilic drugs. Prior research indicated the advantages of triggered release in drug delivery systems. As a specific example, a series of trans-2-aminocyclohexanol based lipids (flipids) have been reported to illustrate a promising strategy to render pH-triggered drug delivery systems: pH-triggered conformational switch. Based on the foregoing, we hypothesize that incorporation of lipids with a pH-sensitive conformational switch and a long-saturated lipid tail can improve the anticancer activities of stealth liposomes. In this study, six new flipids with C-16 saturated hydrocarbon tails were designed. Such lipids were synthesized with high yields by introducing a catalyst (Copper (II) tetrafluoroborate) at a key step of the synthetic scheme. pH-sensitive liposomes (fliposomes) composed of flipids were prepared and loaded with the anticancer drug doxorubicin with high encapsulation efficiency. The physicochemical properties of doxorubicin-loaded fliposomes were characterized and their pH-dependent leakage were investigated. The results showed that among all groups fliposomes containing the C-16 trans-2-morpholylcyclohexanol-based flipid (Mor-C16) exhibited the largest increase of release as the pH dropped form pH 7.4 to 6.0, indicating its good potential of serving as a component in pH-triggered drug delivery systems. Three-dimensional multicellular spheroids (3D MCS) are self-assembled microscale tissue analogs in vitro. They better mimic the native and complex tumor microenvironment than the conventional two-dimensional cell culture systems. In this dissertation study, 3D MCS of six different human cancer cells were successfully cultured and their growing conditions were optimized to obtain 3D MCS of tight structure and reproducible size. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells as well as acidic inside pH based on confocal microscopic imaging studies. The penetration of doxorubicin-loaded Mor-C16 fliposomes into 3D MCS was imaged by confocal microscopy in comparison to doxorubicin-loaded non pH-sensitive liposomes and free doxorubicin. The anticancer activities of doxorubicin-loaded Mor-C16 fliposomes against 3D MCS of three different cell lines was also evaluated by cell viability. Both the fliposome and the non pH-sensitive liposome formulations more efficiently penetrated into two of the three types of 3D MCS compared to free doxorubicin after 4h drug exposure. However, doxorubicin-loaded Mor-C16 fliposome imposed higher cytotoxicity to all three types of 3D MCS compared to doxorubicin-loaded non pH-sensitive liposome over 72 h drug exposure. Taken together, we propose that fliposomes achieved superior activity against 3D MCS by efficient penetration into 3D MCS, followed by enhanced release of the anticancer drug doxorubicin.

Protein sequences contain the information in order for a protein to fold to a unique compact, three-dimensional native structure. The forces that drive protein structures to form compact folds are largely dominated by burial of hydrophobic amino acids, which results in non-specific packing of amino acid side-chains. The knob-socket model attempts to organize side-chain packing into tetrahedral packing motifs. This tetrahedral motif is characterized with a three residues on the same secondary structure forming the base of the tetrahedron packing with a side-chain from a separate secondary structure. The base of the motif is termed the socket, and the other side-chain is called the knob. Here, we focus on extending the knob-socket model to understand tertiary and quaternary structure. First, single knobs sometimes pack into more than one socket in real structures. We focus on understanding the topology and amino acid preferences of these tertiary packing surfaces. The main results from the study of tertiary packing surfaces is that they have a preferred handedness, some interactions are ancillary to the packing interaction, there are specific amino preferences for specific positions in packing surfaces, and there is no relationship between side-chain rotamer of the knob packing into the tertiary packing surface. Next, we examine the application of the knob-socket to irregular and mixed packing in protein structure. The main conclusions from these efforts show canonical packing modes between secondary structures and highlight the important of coil secondary structure in providing many of the knobs for packing. Third, we investigate protein quaternary structure with a clique analysis of side-chain interactions. We identify a possible pseudo knob-socket interaction, and compare knob-socket interactions between tertiary and quaternary structure. Lastly, we discuss the workflow used in CASP12 to predict side-chain contacts and atomic coordinates of proteins.

Proteins are molecular machines of life in the truest sense. Being the expressors of genotype, proteins have been a focus in structural biology. Since the first characterization and structure determination of protein molecule more than half a century ago1, our understanding of protein structure is improving only incrementally. While computational analysis and experimental techniques have helped scientist view the structural features of proteins, our concepts about protein folding remain at the level of simple hydrophobic interactions packing side-chain at the core of the protein. Furthermore, because the rate of genome sequencing is far more rapid than protein structure characterization, much more needs to be achieved in the field of structural biology. As a step in this direction, my dissertation research uses computational analysis and experimental techniques to elucidate the fine structural features of the tertiary packing in proteins. With these set of studies, the knowledge of the field of structural biology extends to the fine details of higher order protein structure.

Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors.

Caractériser la structure tridimensionnelle des protéines avec les structures secondaires classiques est assez pauvre structurellement. Nous avons donc développé une nouvelle méthodologie pour concevoir des séries de petits prototypes moyens nommés Blocs Protéiques (BPs) qui permettent une bonne approximation des structures protéiques. L'analyse de la spécificité des blocs protéiques a montré leur stabilité et leur spécificité sur le plan structural. Le choix final du nombre de BPs est associé a une prédiction locale correcte.
Cette prédiction se base avec une méthode bayésienne qui permet de comprendre l'importance des acides aminés de maniè;re simple. Pour améliorer cette prédiction, nous nous sommes bases sur deux concepts : (i) 1 repliement local -> n séquences et (ii) 1 séquence -> n repliements. Le premier concept signifie que plusieurs types de séquences peuvent être associes a la même structure et le second qu'une séquence peut-être associée a plusieurs type de repliements. Ces deux aspects sont développés en se basant sur la recherche d'un indice de fiabilité lie a la prédiction locale, pour trouver des zones de fortes probabilités. Certains mots, i.e. successions de blocs protéiques apparaissent plus fréquemment que d'autres. Nous avons donc défini au mieux quelle est l'architecture de ces successions, les liens existants entre ces différents mots.
Du fait de cette redondance qui peut apparaìtre dans la structure protéique, une méthode de compactage qui permet d'associer des structures structurellement proches sur le plan local a été mise au point. Cette approche appelée "protéine hybride" de conception simple permet de catégoriser en classes "structurellement dépendantes" l'ensemble des structures de la base de données protéiques. Cette approche, en plus du compactage, peut être utilisée dans une optique différente, celle de la recherche d'homologie structurale et de la caractérisation des dépendances entre structures et séquences.

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic as well as potential therapeutic agent. The Proteolysis Targeting Chimera (PROTAC) is a bi-functional small molecule containing a ligand and proteolysis inducer. PROTAC recruits the target protein to proteolysis machinery and elicits proteolysis. Thus far, a number of PROTAC have been prepared and demonstrated to effectively induce the degradation of targeted protein in cultured cells, validating PROTAC as a useful research tool. In the present study, PROTACs based on apigenin was prepared and demonstrated to induce the degradation of AHR, providing the proof of concept. To improve activity, a synthetic structure, CH-223191, was optimized for antagonistic activity by positional scanning identifying several AHR antagonists. PROTACs based on the optimal structure were prepared and assessed their biological activity. The products and synthetic scheme described hereby will be helpful for the further understanding on AHR biology as well as for developing therapeutic agents targeting AHR.

Un photosensibilisateur est un composé chimique capable de générer, sous l'effet d'une irradiation lumineuse, des espèces réactives de l'oxygène (ROS) et donc d'induire directement ou indirectement l'altération d'autres composés. La rétention sélective de ces molécules, dites photo-activables, par les tissus en prolifération leur confère des applications en thérapie anti-tumorale (Photo-Chimio-Thérapie, PDT). La plupart des photosensibilisateurs sur le marché sont des dérivés structuraux de porphyrines et sont généralement fluorescents. Cette propriété est utilisée pour déterminer leur localisation, tant au niveau systémique que cellulaire. Certains composés sont ainsi utilisés pour le diagnostic par fluorescence de certain cancers (FD). Au niveau intracellulaire, un certain nombre de ces photosensibilisateurs se localisent dans les membranes des vésicules d'endocytose. Ils peuvent ainsi induire, sous irradiation lumineuse, la libération dans le cytosol du contenu de ces vésicules, y compris des molécules exogènes incorporées dans la cellules par endocytose et dont la cible intracellulaire est cytosolique ou nucléique (ADN, protéines, nombreux médicaments...). Ces phénomènes sont à la base d'une approche permettant l'activation de macromolécules, l'Internalisation Photo-Assistée (PCI). Cette méthode potentialise considérablement l'activité biologique d'un très large spectre de molécules d'intérêt.Tant la sélectivité de ces photosensibilisateurs pour les tissus en prolifération que leur localisation intracellulaire sont à mettre en relation avec les propriétés physico-chimiques et biologiques du micro-environnement. Leur interaction avec les lipoprotéines de basse densité (LDL) peut favoriser leur entrée dans les cellules du fait de la surexpression par les cellules néoplasiques des récepteurs aux LDL. Cependant, pour certaines molécules photo-activables, un passage transmembranaire par diffusion passive a été mis en évidence. L'incorporation cellulaire est alors facilitée par l'acidification du pH stromatique. Nous nous sommes donc attaché à déterminer l'importance de tels paramètres. Notre objectif a été de définir des caractéristiques structurales déterminant le comportement cellulaire des photosensibilisateurs.Dans un premier temps, afin d'élucider les mécanismes impliqués, les interactions de photosensibilisateurs avec des LDL et des liposomes (SUV, utilisés comme modèles simples de membranes) ont été étudiées à l'équilibre et de façon dynamique. Trois photosensibilisateurs ont été utilisés : la deuteroporphyrin (DP), une porphyrine dicarboxylique et la phtalocyanine d'aluminium disulfonnée (AlPcS2). Ces études ont été menées en nous attachant tout particulièrement aux effets liés au pH. Il faut noter ici que seuls les composés carboxyliques sont susceptibles de subir une neutralisation, ne serait-ce que partielle, de leurs chaînes latérales dans une gamme de pH correspondant à des valeurs physiologiques. Les données obtenues sur ces systèmes simples nous ont ensuite permis de comprendre la localisation sub-cellulaire des photosensibilisateurs sur une lignée humaine de fibroblastes. Enfin, bien que les LDL soient des vecteurs importants des photosensibilisateurs et facilitent leur entrée dans les cellules, la localisation sub-cellulaire semble être directement liée à la dynamique du transfert des photosensibilisateurs par des membranes. En conclusion, les paramètres physico-chimiques déterminés en solutions sont des outils efficaces pour concevoir des photosensibilisateurs, prédire leur capacité d'incorporation cellulaire ainsi que leur localisation sub-cellulaire.

L'objectif de cette thèse consiste à inhiber la voie de signalisation liée aux protéines Ras, très fréquemment impliquée dans les tumeurs humaines, en concevant des inhibiteurs d'interactions protéine-protéine. Au sein de cette voie, la protéine RasGAP joue un rôle particulier, à la fois régulateur négatif et effecteur de Ras. La cible thérapeutique constituée par le domaine SH3 de la protéine RasGAP avait déjà été identifiée par plusieurs équipes (Tocqué et al., 1997). Plus récemment, notre laboratoire a complété ces travaux par l'identification des protéines Aurora comme partenaires de RasGAP-SH3. En collaboration avec Aptanomics, en mettant en oeuvre la technique des aptamères peptidiques, nous avons apporté une nouvelle validation de cette cible : nous avons obtenu par un crible double-hybride de nouvelles protéines synthétiques (aptamères) interagissant spécifiquement avec le domaine SH3 de RasGAP, et dont l'expression dans des cellules tumorales provoque une diminution de la capacité à former des colonies et de la viabilité cellulaire. Afin d'amorcer une démarche de conception rationnelle d'inhibiteurs de ce SH3, nous avons synthétisé les peptides exposés à la surface de ces aptamères et responsables de leur interaction avec RasGAP-SH3. Nous avons ensuite mesuré l'affinité de ces peptides pour RasGAP-SH3 par anisotropie de fluorescence. Nous avons ainsi obtenu un peptide cyclique dont l'affinité pour le domaine SH3 est de l'ordre de quelques centaines de nanomolaire, et dont nous avons déterminé l'empreinte sur ce domaine enrichi en 15N par RMN, en nous appuyant sur la structure du domaine, déjà résolue au laboratoire (Yang et al., 1994). Les données structurales que nous avons obtenues devraient permettre, dans un court délai, de proposer des modifications de ces peptides, afin d'augmenter l'affinité de nos inhibiteurs et de les vectoriser pour leur conférer une activité sur cellules tumorales en culture. Enfin, ces peptides, rendus fluorescents par leur couplage à un fluorophore, vont être utilisés comme ligands de référence dans un crible de chimiothèque à haut débit, afin de découvrir de petites molécules inhibitrices du domaine SH3 de RasGAP.

Molecular dynamics (MD) is a powerful tool that can be applied to protein folding and protein structure. MD allows for the calculation of movement, and final position, of atoms in a biomolecule. These movements can be used to investigate the pathways that allow proteins to fold into energetically favorable structures. While MD is very useful, it still has its limitations. Most notable, computing power and time are of constant concern. Protein structure is inherently important due to the direct link between the structure of a protein and its function. One of the four levels of protein structure, the secondary structure, is the first level to accommodate for the three-dimensional shape of a protein. The main driving force behind secondary structure is hydrogen bonding, which occurs between the carboxyl oxygen and the amine hydrogen of the backbone of a peptide. Determining a greater link between hydrogen bond patterns and types of secondary structure can provide more insight on how proteins fold. Because molecular dynamics allows for an atomic level view of the dynamics behind protein folding/unfolding, it becomes very useful in observing the effects of particular hydrogen bond patterns on the folding pathway and final structure formed of a protein. Using molecular dynamic simulations, a series of experiments in an attempt to alter structure, hydrogen bonding, and folding patterns, can be performed. This information can be used to better understand the driving force of secondary structure, and use the knowledge gained to manipulate these simulations to force folding events, and with that, desired secondary structure features.

La motivation première de mes travaux de recherche est de combiner des approches expérimentales et théoriques dans le domaine de la chimie physique pour atteindre une meilleure compréhension des phénomènes à l'échelle atomique. Mes travaux en cours traitent de systèmes d'intérêt biologique concernant les processus membranaires et des phénomènes accessibles par des méthodes de nanomanipulation. Les problèmes de la biophysique et biochimie sont au c?ur de mes recherches. J'ai effectué des simulations complexes de protéines membranaires dans une bicouche lipidique qui se sont montrées tout à fait complémentaires et révélatrices par rapport aux études expérimentales de biologie structurale. Une récente collaboration exploitant cette complémentarité a donné lieu à une publication dans la revue Nature en début 2009. [[i]] Les travaux récents visent à développer des approches combinant la réalité virtuelle avec les simulations moléculaires. [[ii]] Les systèmes biologiques étudiés présentent à la fois un intérêt physico-chimique, biologique et médical et peuvent atteindre un grand nombre d'atomes. En parallèle, je mène un travail de fond sur les méthodes de simulation et des approches novatrices. ----------------------- [[i]] N. Bocquet, H. Nury, M. Baaden, C. Le Poupon, J.P. Changeux, M. Delarue et P.J. Corringer: "X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation", 2009, Nature, 457, 111-114. [[ii]] O. Delalande, N. Férey, G. Grasseau et M. Baaden : "Complex Molecular Assemblies at hand via Interactive Simulations", 2009, J. Comput. Chem., 30, 2009, 2375-2387.

Ce manuscrit présente une étude fonctionnelle et structurale du cytochrome P450 2J2 humain (CYP2J2), enzyme exprimée dans les tissus cardiovasculaires, dont les rôles biologiques sont mal connus. En utilisant la terfénadone comme base structurale, qui est un composé oxydé régiosélectivement par le CYP2J2, plusieurs composés ont été synthétisés se sont révélés être des inhibiteurs affins et sélectifs du CYP2J2. En particulier, un inhibiteur très affin et compétitif (Ki = 160 nM) et deux substrats suicides efficaces du CYP2J2 (kinact/Ki 3000 L/mol/s) ont été mis en évidence. L'étude de l'oxydation de ces composés par le CYP2J2 a révélé une régiosélectivité surprenante, en faveur d'une position chimiquement moins réactive vis-à-vis des oxydations. La caractérisation du site actif du CYP2J2 et l'identification de résidus importants pour la reconnaissance des dérivés de terfénadone a pu être réalisée en construisant un modèle par homologie 3D de cette enzyme et par le docking de certains dérivés dans le site actif du CYP2J2. Enfin, une étude préliminaire des rôles biologiques possibles du CYP2J2 a été réalisée en étudiant les effets inhibiteur de ce P450. En conclusion, ce travail a permis de caractériser les premiers outils biochimiques d'étude des rôles biologiques du CYP2J2, de proposer une première topologie du site actif du CYP2J2, et d'affiner les rôles biologiques du CYP2J2.

En 1940, le biochimiste anglais Donald Woods propose une explication du mode d'action des nouveaux sulfamides antibactériens : l'inhibition compétitive. Son collègue Paul Fildes fonde sur cette base une nouvelle approche de la chimiothérapie, revendiquée comme rationnelle, un programme pour la recherche de nouveaux médicaments. Cette thèse explore l'impact de la théorie des antimétabolites, comme elle sera appelée, dans la recherche biochimique et pharmaceutique. La première partie retrace son élaboration dans le contexte de l'école de biochimie anglaise, puis sa reprise aux États-Unis à la suite de travaux menés en parallèle sur les vitamines. La seconde partie est consacrée au développement de deux programmes de recherche distincts dédiés aux antimétabolites, illustrant les modalités et fortunes divergentes d'appropriation de cette approche rationnelle. Le premier est une collaboration modeste entre le biochimiste Henry McIlwain et la firme pharmaceutique Glaxo pendant la guerre au Royaume-Uni. Le second consiste en la mise en place du programme de George Hitchings et Gertrude Elion chez Burroughs Wellcome aux États-Unis, souvent considéré comme l'origine du rational drug design actuel. La théorie des antimétabolites correspond aussi bien à l'ambition d'obtenir des chimiothérapies spécifiques qu'à un ensemble de pratiques dans le quotidien du laboratoire
In 1940, the British biochemist Donald Woods put forward an explanation of the mode of action of the new antibacterial sulfa drugs, competitive inhibition. His colleague, Paul Fildes, developed this work into a new approach to chemotherapy, which he qualified as a rational programme for drug discovery. This dissertation explores the impact of the theory of antimetabolites, as it came to be known, in biochemical and pharmaceutical research. The first part traces its development in the context of the British school of biochemistry and its further expansion in the United States following parallel research on vitamins. The second part deals with the construction of two distinct research programmes dedicated to antimetabolites, each one illustrating a different way of following this rational approach and their varying consequences. The first one is a modest collaboration between the biochemist Henry McIlwain and the Glaxo pharmaceutical company during the war in the United Kingdom. The second one corresponds to the establishment of George Hitchings' and Gertrude Elion's programme at Burroughs Wellcome in the United States, often considered as the origin of today's rational drug design. The theory of antimetabolites simultaneously embodied both the ambition of attaining specific chemotherapies, and a set of practices in day-to-day laboratory work

Dans la perspective de trouver de nouvelles thérapies dans le traitement du diabète de type II, non insulino-dépendant, le Glucagon-Like Peptide-1 (GLP-1) constitue un excellent candidat. Si son mécanisme d'action est bien connu, il reste toutefois à résoudre de grands problèmes fondamentaux, avant de le substituer aux molécules utilisées pour une telle pathologie. En particulier, la compréhension de la liaison du GLP-1 à son récepteur demeure un point crucial. Une meilleure connaissance des structures du ligand et du récepteur sont nécessaires. De plus, ce peptide ne peut être utilisé dans sa forme native, dû à une inactivation rapide par les protéases.

Pour essayer d'augmenter la stabilité du peptide, et en tenant compte des positions clés définies dans la littérature, plusieurs analogues du GLP-1-(7-37) sont conçus, et synthétisés. Ils possèdent principalement des pharmacomodulations au niveau de la partie N-terminale. Des substitutions sont également réalisées dans la partie centrale du peptide, permettant de vérifier certaines hypothèses concernant sa conformation. Considérant les résultats de liaison et d'efficacité in vitro, certains analogues sont sélectionnés pour des études in vivo d'activité et de stabilité métabolique. Le [a8,desR36]GLP-1-(7-37) se distingue des autres tant par sa grande stabilité que son efficacité, supérieure à la molécule native. Ce composé est en phase de développement pré-clinique.

Parallèlement, la conformation de chaque analogue est étudiée (CD, IR) et ainsi, confrontée aux résultats in vitro, il est possible de proposer une conformation bioactive.

Enfin, pour appréhender plus en avant les mécanismes de liaison du peptide avec son récepteur spécifique, la modélisation moléculaire du récepteur fait ressortir quelques hypothèses quant à la localisation probable de l'interaction hormone-récepteur. Des analyses biophysiques et la synthèse de fragments du récepteur, ont permis d'étayer de telles hypothèses.

In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens. HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert. Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively. In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable. Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease. This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.

Les aminoglycosides, des dérivés aminés de saccharides, interfèrent avec le mécanisme de synthèse des protéines chez les bactéries en se fixant au site de décodage de l'ARN de transfert aminoacylé (site A) situé en 3' de l'ARN ribosomique 16S. Au cours de ce travail de thèse, les structures de trois complexes entre des fragments d'ARN incorporant le site A et les aminoglycosides paromomycine, tobramycine et généticine, ont été résolues par cristallographie aux rayons X à 2,40-2,54 Å. L'analyse des structures montre que la reconnaissance et la fixation spécifiques des aminoglycosides au site A font intervenir de nombreuses liaisons hydrogène directes et pontées par des molécules d'eau. Dans ces structures, la partie néamine commune aux aminoglycosides (cycles I et II) s'intercale dans l'hélice de manière similaire : le cycle I (non plan) forme une pseudo paire de bases avec l'adénine 1408 ; la néamine oblige les adénines 1492 et 1493 à pointer hors de l'hélice. La comparaison des structures 3D de ces trois complexes offre des explications moléculaires aux différents résultats de biochimie et de microbiologie, ainsi qu'à certains phénomènes de résistances et de toxicités. Les conformations du site A et des aminoglycosides au sein de ces complexes sont similaires à celles du site A et de la paromomycine au sein de la sous-unité ribosomique 30S. Ainsi, la stratégie développée permet une description des interactions et des modes de fonctionnement des aminoglycosides proche du contexte naturel mais plus précise, essentielle à notre connaissance du système ARN/aminoglycoside. De ces résultats découlent des lignes directrices laissant envisager sous un jour nouveau la conception d'antibiotiques moins sujets aux résistances et moins toxiques.

La RMN en phase solide est une méthode non négligeable lors de l'étude d'échantillons biologiques. Nous avons ainsi pu étudier le peptidoglycane et les acides téichoïques, composants essentiels de la paroi cellulaire bactérienne. Nous nous sommes tout particulièrement intéressés à leur organisation, à leur flexibilité et à leur interaction avec les cations. Nous avons également étudié les interactions entre les acides téichoïques et une protéine responsable de la viru-lence des pneumocoques. Cet exemple illustre parfaitement le manque de sensi-bilité des expériences de RMN ainsi que les limites des techniques actuelles de recouplage. Nous avons alors mis en évidence un phénomène permettant d'accélérer l'acquisition des spectres. Nous avons aussi travaillé à l'amélioration des séquences de recouplage PAR et PAIN-CP. Finalement, nous avons compa-ré des méthodes d'édition spectrale appliquées au bois, très étudié dès les débuts de la RMN en phase solide.

Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.

Les grilles de calcul sont une nouvelle Technologie de l'Information permettant la collecte et le partage de l'information, la mise en réseau d'experts et la mobilisation de ressources en routine ou en urgence. Elles ouvrent de nouvelles perspectives de réduction des coûts et d'accélération de la recherche in silico de médicaments contre les maladies négligées et émergentes. Dans ce contexte, la première partie de la thèse a porté sur la conception de services bio-informatiques sur grille. Ils facilitent le déploiement et la mise à jour sur la grille RUGBI de logiciels et de bases de données. La seconde partie a vu le déploiement d'expériences de criblage virtuel à haut débit sur l'infrastructure de grille EGEE. Les expériences ont démontré que les grilles collaboratives ont la capacité à mobiliser d'importantes ressources de calcul dans des buts bien définis pendant une période de temps significative, et qu'elles produisent des résultats biologiques pertinents.

A travers cette thèse, nous montrons l'importance de la modélisation et de la coopération de métaheuristiques pour la résolution de problèmes réels en bioinformatique. Pour ce faire, deux problèmes ont été étudiés : le premier dans le domaine de la protéomique pour l'identification de protéines à partir de données spectrales et le second dans le domaine de l'analyse structurale de molécules pour le problème du docking moléculaire flexible. Ainsi, pour le premier problème, un nouveau modèle basé sur une comparaison directe des bases de données protéiques avec les données expérimentales brutes a été mise en place. L'approche associée a été intégrée au sein d'un moteur d'identification par empreinte de masse peptide appelé ASCQ_ME. Ce modèle d'identification a permis ensuite de proposer et de valider une modélisation pour le problème de " de novo protein sequencing " qui consiste à retrouver la séquence d'une protéine à partir seulement des données expérimentales. Il s'agit d'un modèle en trois étapes appelé SSO pour " Sequence ", " Shape " et " Order ". Après une étude de chacune de ces étapes, SSO a été implémenté et testé à travers trois métaheuristiques collaborant de manière séquentielle. Pour le second problème, une étude des nouvelles modélisations multi-objectives a été menée et a conduit à la définition d'un ensemble de huit modèles différents testés à l'aide d'algorithmes génétiques multi-objectifs parallèles. Une douzaine de configuration d'opérateurs génétiques ont été testé afin de mettre en évidence l'efficacité de l'hybridation des algorithmes génétiques avec des recherches locales. Pour chacune des parties, l'implémentation et la mise en place des collaborations fut possible grâce à la plateforme ParadisEO et notamment grâce à mes contributions à la partie ParadisEO-MO dédiée aux métaheuristiques à base de solution unique. L'ensemble de ces travaux a été soutenu par le PPF BioInformatique de l'Université des Sciences et Technologies de Lille et le projet ANR Dock.

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens.

In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first-generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky moieties of early inhibitors create potential clashes with the a-b loop of Insert I of non-susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections.

In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA.

Dissertation

Thrombosis is associated with the occlusion of a blood vessel and can be triggered by a number of types of injury, such as the rupture of an atherosclerotic plaque on the artery wall, changes in blood composition, or blood stasis. The resulting thrombosis can cause major diseases such as myocardial infarction, stroke, and venous thromboembolic disorders that, collectively, account for the most common cause of death in the developed world. Anticoagulants are used to treat and prevent these thrombotic diseases in a number of clinical and surgical settings. Although commonly prescribed, currently approved anticoagulants have a major limitation of severe drug-induced bleeding that contributes to the high levels of morbidity and mortality associated with use. The "holy grail" for antithrombotic therapy is to identify a drug that inhibits thrombus formation without promoting bleeding. Understanding the differences between thrombosis and hemostasis in the vascular system is critical to developing these safe and effective anticoagulants, as this depends on striking the correct balance between inhibiting thrombus formation (efficacy) and reducing the risk of severe bleeding (safety). While it is commonly thought that the same factors play a similar role in hemostasis and thrombosis, recent evidence points to differing functions for FXI and FXII in each of these settings. Importantly, these factors seem to contribute to pathological thrombus formation without being involved in normal hemostasis.

The overall goal of this project was to evaluate the inhibition of the intrinsic pathway of coagulation as a potential anticoagulant strategy utilizing the aptamer platform. Aptamers are short, highly structured nucleic acids that act as antagonists by binding to large surface areas on their target protein and thus tend to inhibit protein-protein interactions. High affinity binding aptamers have been isolated that specifically target a diverse range of proteins, including transcription factors, proteases, viral proteins, and growth factors, as well as other coagulation factors. As synthetic molecules, aptamers have a small molecular weight, are highly amenable to modifications that can control their bioavailability, and have not been found to elicit an immune response, thus making them ideal drug candidates. Importantly, aptamers can be rapidly and effectively reversed with either a sequence specific antidote that recognizes the primary sequence of the aptamer or a universal antidote that binds to their backbone and reverses all aptamer activity independent of sequence. This ability lends itself well to their therapeutic application in coagulation, as rapid reversal of a drug upon the onset of bleeding is a key property for increasing the safety of this class of drugs.

Aptamers targeting FXI/FXIa and FXII/FXIIa were isolated in two separate SELEX (systematic evolution of ligands by exponential enrichment) procedures: the FXII aptamer was isolated in a convergent SELEX approach and the FXIa aptamer was isolated from a purified protein selection. In both processes, 2'fluoropyrimindine modified RNA with a 40-nucleotide random region was incubated with either the plasma proteome (in initial rounds of the convergent SELEX) or the purified protein target (FXII or FXIa). The nucleic acids that did not bind to the target were separated from those that bound, and these molecules were then amplified to generate an enriched pool with increased binding affinity for the target. This process was repeated under increasingly stringent conditions to isolate the aptamer that bound with the highest affinity to the purified target protein. Utilizing biochemical and in vitro coagulation assays, specific, high-affinity binding and functional anticoagulant aptamers were identified for both protein targets, and the mechanism of anticoagulation was ascertained for each aptamer.

Overall, both aptamers bound to an exosite on their target protein that was able to inhibit downstream activation of the next protein in the coagulation cascade. In order to specifically examine aptamer effects on several parameters of thrombin generation, a new assay was developed and fully characterized using aptamer anticoagulants targeting other coagulation factors. Aptamer inhibition of both FXI and FXII was able to decrease thrombin generation in human plasma. However, limited cross-reactivity in other animal species by both aptamers hindered our ability to assess aptamer inhibition in an in vivo setting. Moving forward, screening aptamers against a larger selection of animal plasmas will hopefully allow us to identify an animal species in which we can analyze aptamer inhibition of the intrinsic pathway for effectiveness and safety in inhibiting thrombosis. The further characterization and use of these aptamers in plasma and blood based settings will allow us to study the diverging functions of the intrinsic pathway in thrombosis and hemostasis.

A critical need exists for safe and effective anticoagulants to treat and prevent numerous thrombotic procedures and diseases. An ideal anticoagulant is one that strikes the correct balance between inhibiting thrombus formation and reducing drug-induced bleeding. Inhibition or depletion of factors XI and XII of the intrinsic pathway of coagulation have shown reduced thrombus formation without interruption of normal hemostasis in several models of thrombosis. By developing novel RNA aptamer anticoagulants to these factors, we have set the stage for evaluating the net therapeutic benefit of intrinsic pathway inhibition to effectively control coagulation, manage thrombosis, and improve patient outcome. As well as developing a safe anticoagulation, these agents can lead to important biological discoveries concerning the fundamental difference between hemostasis and thrombosis.

Dissertation

abstract: The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4.
Dissertation/Thesis
Ph.D. Chemistry 2013

Orientação: Isabel Ribeiro
Apesar dos hidratos de carbono representarem apenas 1% do peso corporal, são essencialmente importantes nas reações químicas que fornecem energia às células, principalmente ao cérebro. A glicose é o monossacarídeo com maior relevância para obtenção de energia. As outras oses provenientes dos hidratos de carbono vão-se integrar no metabolismo da glicose. Para manter a glicémia dentro do intervalo de valores de referência, há um sistema de regulação endócrino, do qual a insulina e o glucagon desempenham um papel predominante. A hipoglicémia define-se como um estado metabólico caracterizado por níveis de glicémia inferiores a 55 mg/dL, acompanhada de manifestações clínicas de intensidade e expressão variáveis, que refletem sintomas como a ansiedade, palpitações, tremores, défice cognitivo e coma. A glicose presente no organismo pode ser proveniente da dieta, ou da produção endógena. Deste modo, a hipoglicémia pode resultar de um consumo excessivo de glicose (exercício físico ou aumento de perdas externas) ou de um inadequado aporte de glicose (produção endógena insuficiente ou inanição). A hipoglicémia é uma complicação aguda, muito frequente, que surge como consequência do tratamento da diabetes com insulina e/ou sulfonilureias e, com muito menos frequência, no individuo não diabético. Neste, a hipoglicémia pode ser consequência primária de uma patologia, ou seja, por intervenção direta no metabolismo da glicose, ou consequência secundária de uma patologia, ou seja, por um mecanismo não direto. O estado de jejum ou pós-prandial do individuo quando surge a hipoglicémia também auxilia no diagnóstico diferencial. Para estabelecer o diagnóstico é necessário se verificar a tríade de Whipple: (1) sinais e sintomas compatíveis com hipoglicémia; (2) baixa concentração de glicémia; (3) melhoria dos sintomas após aumento da glicémia. O tratamento da hipoglicémia passa pela administração de hidratos de carbono, nomeadamente sacarose. O presente trabalho tem como objetivo, numa primeira parte, fazer a revisão, do ponto de vista bioquímico, dos mecanismos associados ao metabolismo dos glícidos e da regulação da glicémia que contribuem para a rápida correção da hipoglicémia. Numa segunda parte, pretende-se descreve-se as manifestações clinicas, as várias classificações de hipoglicémia e os mecanismos em cada caso/patologia. Numa terceira parte apresenta-se a abordagem diagnóstica na suspeita de hipoglicémia e qual a terapêutica mais adequada. Por último pretende-se evidenciar a relação da hipoglicémia versus hiperglicemia. Enquanto estados crónicos de hiperglicemia têm sido associados a disfunções de vários órgãos a longo prazo, episódios hipoglicémicos, apesar de pontuais, estão associados, a lesões neurológicas a curto prazo e até morte. Associações cientificas como The American Diabetes Association e The Endocrine Society têm vindo a disponibilizar informação para uma maior compreensão do episódio hipoglicémico, as suas implicações e estratégias para prevenção. Neste sentido, concluiu-se que é essencial identificar o mecanismo que conduziu à hipoglicémia de modo a prevenir a sua recorrência.

La RMN est une méthode de choix pour la détermination de la structure tridimensionnelle des protéines et des acides nucléiques en solution. Cependant, la taille des systèmes que l'on peut étudier actuellement par RMN est limitée. Dans la première partie de ce travail, la structure du répresseur BlaI de la beta-lactamase de B. licheniformis 749/I et son interaction avec l'ADN ont été étudiées par RMN avec des méthodes classiques. Ces résultats ont permis de mieux caractériser la répression des gènes de plusieurs mécanismes de résistance aux antibiotiques, incluant la résistance à la méthicilline de la souche pathogène S. aureus. Le deuxième volet de ce travail concerne l'implémentation de filtres Hadamard qui augmentent la résolution des spectres dans certaines expériences de RMN multidimensionnelle. Ces filtres permettent de séparer les pics de corrélation des protéines et des acides nucléiques selon le type d'acide aminé et le type de base, respectivement. Ces développements ouvrent de nouveaux horizons vers l'étude de macromolécules biologiques de plus grosse taille par RMN.