The sirtuins are a family of NAD⁺-dependent deacetylase enzymes which are implicated in various illnesses including cancer and neurodegenerative diseases. Part I of this thesis describes the synthesis and biological evaluation of inhibitors of the SIRT1 and SIRT2 isoforms of this important family of enzymes. Chapter 1 gives an overview of sirtuin biology and the physiological roles of these enzymes. In particular the link between SIRT1 and cancer and SIRT2 and its role in the onset of neurodegenerative diseases is discussed. A review of the most potent and selective inhibitors of SIRT1 and SIRT2 is given including an introduction to the tenovin and cambinol classes of inhibitor. Chapter 2 describes various issues relating to the structure of the important chemical tool tenovin-6. The synthesis of analogues to improve the solubility, determine the preferred conformation and verify the products of metabolism of tenovin-6 is presented including their evaluation by in vitro and in cell methods. Part II of this chapter reports the design and use of a ¹H NMR method used to monitor the sirtuin-mediated deacetylation reaction. This was particularly relevant due to concerns raised about the possibility of false positive results obtained with the commercially available assay kit commonly used by the sirtuin community. This new ¹H NMR method was used to validate the inhibition of SIRT2 by tenovin-6. Chapter 3 describes the parallel synthesis and evaluation of tenovin analogues as inhibitors of SIRT1 and SIRT2. This study identified that replacement of the t-butyl substituent of tenovin-6 with the 3,5-dihalogen-4-alkoxy substitution pattern led to a variety of analogues having SIRT2 selectivity. As well as the collection of valuable SAR data, in cell data is also presented for the analogues. Chapter 4 provides attempts to rationalise the SAR data collected in Chapters 2 and 3 through a computational study. The molecular docking software GOLD was used to predict the binding site of the tenovin scaffold and hence rationalise the observed potencies of various analogues. Chapter 5 reports the synthesis and biological evaluation of triazole and cambinol analogues as SIRT1 and SIRT2 inhibitors. Part I details the synthesis and in vitro testing of a series of ring constrained tenovin analogues based on the 1,4-disubstituted triazole using click chemistry. A series of 1,5-disubstituted analogues were also synthesised. Part II describes the synthesis of S-alkylated cambinol analogues and the effect of N3-methylation upon activity and selectivity towards SIRT1. Part II of this thesis details the synthesis and biological testing of novel potent allosteric inhibitors of RmlA. RmlA is the first enzyme in the L-rhamnose biosynthetic pathway in bacteria. L-rhamnose is an important component of the bacterial cell wall and as such RmlA is therefore an important target in the discovery of novel anti-bacterial compounds. Chapter 7 provides an overview of the RmlA enzyme including its role in L-rhamnose biosynthesis and why it is an attractive target for anti-bacterial drug discovery. No small molecule inhibitors of RmlA have been reported previously. Chapter 8 describes the design and synthesis of pyrimidine-2,4-dione analogues as novel allosteric inhibitors of RmlA. SAR data is generated and rationalised by X-ray crystallographic techniques to study the structures of complexes of RmlA with various analogues. Analogues were also tested for their ability to inhibit the growth of the important human pathogen Mycobacterium tuberculosis.

Les moteurs moléculaires sont des protéines capables de produire une force : elles transforment l'énergie chimique de l'hydrolyse de l'ATP en énergie mécanique. Cette thèse se focalise sur l'étude d'une famille de moteurs moléculaires, les myosines, qui se déplacent le long des filaments d'actine et assurent d'importantes fonctions cellulaires.La myosine VI est une myosine très particulière car elle est la seule à se déplacer vers l'extrémité négative des filaments d'actine. Elle est produite dans la cellule sous forme auto-inhibée, inactive. Dans la cellule, son activité est également régulée par plusieurs protéines interagissant avec la queue C-terminale de la myosine VI. Ces protéines, présentes à des endroits précis de la cellule, recrutent la myosine VI et dictent l'action qu'elle doit effectuer. Des analyses de SAXS, de dispersion de la lumière, de microscopie, d'interaction et de mutagénèse ont permis de mieux comprendre le mécanisme régulant l'adoption de l'état auto-inhibé, ainsi que son activation par le calcium. L'interaction avec différents partenaires a été caractérisée. GIPC1, le partenaire le plus étudié, promeut de façon indirecte la dimérisation et l'activation de la myosine VI.Pendant ma thèse, j'ai également été impliqué dans deux autres projets qui s'inscrivent dans la logique du projet de thèse et qui ont mené à la publication de quatre articles. Deux chapitres, plus brefs, sont donc dédiés à ces projets. Le deuxième chapitre porte sur la régulation de l'activité de la myosine VII par ses partenaires cellulaires. Finalement, le troisième chapitre est dédié à l'étude de la modification allostérique de l'activité des myosines par des petites molécules. / Molecular motors are essential agents of force production in the cells: they convert the chemical energy released by the hydrolysis of ATP into mechanical work. This thesis focuses on myosins, a family of molecular motors responsible for actin-based motility. Myosin VI is unique among all of the myosin superfamily members in that it moves in the opposite direction of all other known myosins. Previous work revealed myosin VI tail ability to fold back, constituting a potential auto-inhibited state that prevents motor activity. Several myosin VI partners, binding to the C-terminal tail of the myosin, have been identified and shown to recruit the motor for different functions. In the first chapter of this thesis, the mechanism allowing the regulation of myosin VI activity has been studied using biochemical and biophysical analysis (SAXS, light scattering, microscopy, binding assays and mutagenesis). GIPC1, the most studied myosin VI partners, promotes myosin dimerization and activation. During my PhD, I have been also involved in two other projects, in line with my thesis project, that have led to the publication of four articles. Two shorter chapters are therefore devoted to these projects. The second chapter of my thesis explores myosin VII activity regulation by its cellular partners. Finally, the third chapter is devoted to the allosteric regulation of myosins activity by small molecules.

In 1996, scientists discovered a connection between the gene for the human protein frataxin (FXN) and the neurodegenerative disease Friedreich’s ataxia (FRDA). Decreased FXN levels result in a variety of aberrant phenotypes including loss of activity for iron-sulfur containing enzymes, mitochondrial iron accumulation, and susceptibility to oxidative stress. These symptoms are the primary focus of current therapeutic efforts. In contrast our group is pursuing an alternate strategy of first defining FXN function at a molecular level then using this information to identify small molecule functional replacements. Recently, our group has discovered that FXN functions as an allosteric activator for the human Fe-S cluster assembly complex. The work presented here helps to further define molecular details of FXN activation and explain how FRDA missense mutants are functionally compromised. First, the FRDA missense mutants L182H and L182F were investigated. Unlike other characterized FRDA missense mutants, the L182F variant was not compromised in its ability to bind and activate the Fe-S assembly complex. The L182H variant exhibited an altered circular dichroism signature; suggesting a change in secondary structure relative to native FXN, and rapidly degraded. Together these studies suggest that L182 variants are less stable than native FXN and are likely prone to degradation in FRDA patients. Second, as a regulatory role of FXN suggests that its function is likely controlled by environmental stimuli, different maturation forms of FXN as well as post-translational modification mimics were tested as mechanisms to control FXN regulation. Here experiments were designed to test if a larger polypeptide form of FXN represents a functional form of the protein. Kinetic and analytical ultracentrifugation studies revealed a complex heterogeneous mixture of species some of which can activate the Fe-S assembly complex. A previously identified acetylation site was also tested using mutants that mimic acetylation. These mutants had little effect on the ability of FXN to bind and activate the assembly complex. Third, mutagenesis experiments were designed in which the FXN surface residues were replaced with alanine and the resulting variants were tested in binding and activity assays. These experiments revealed a localized “hot-spot” on the surface of FXN that suggests small cyclic peptide mimics might be able to replace FXN and function as FRDA therapeutics. Unexpectedly, one of the FXN variants exhibited significantly tighter binding and could have relevance for therapeutic development.

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS). / Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

No description available.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains. T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other. / Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first reaction in the shikimate pathway, leading to the biosynthesis of aromatic compounds including the aromatic amino acids. The catalytic activity of DAH7PS is regulated through feedback inhibition and is the major control point for the pathway. DAH7PSs are divided into two families, type I and type II, based on molecular weight and amino acid sequence. Type I DAH7PSs can be further divided based on sequence similarity. All DAH7PS enzymes with their crystal structures solved share a basic (β/α)₈-barrel fold in which the key catalytic components are housed. Furthermore, all structurally characterised DAH7PSs, except Pyrococcus furiosus DAH7PS (PfuDAH7PS) and Aeropyrum pernix DAH7PS, have recruited extra structural motifs that are implicated in allosteric regulation. However, there are significant differences in the additional structural elements. This thesis investigates the hypothesis that the diverse regulation strategies for controlling DAH7PS activity have evolved by domain recruitment, whereby regulatory domains have been added to the catalytic barrel. Chapter 2 describes the functional characterisation of the type Iβ Thermotoga maritima DAH7PS (TmaDAH7PS), and the exploration of its response to inhibitors. The catalytic activity of TmaDAH7PS was found to be substantially inhibited by tyrosine (Tyr) and to a lesser extent, phenylalanine (Phe). The putative regulatory domain previously identified as a ferredoxin-like domain was recognised as an aspartate kinase-chorismate-mutase-tyrA (prephenate dehydrogenase) or ACT domain. Chapter 3 describes the characterisation of TmaDAH7PS with the N-terminal domain removed. The truncated enzyme was found to be more catalytically active than wild-type TmaDAH7PS and insensitive to inhibition by the aromatic amino acids, Tyr, Phe and tryptophan. Apart from the truncation of the ACT domain, the crystal structure of truncTmaDAH7PS showed no major changes to the monomer structure when compared to wild-type TmaDAH7PS. However, truncTmaDAH7PS crystallises as a dimer, unlike wild-type TmaDAH7PS. In Chapter 4, the solution of the crystal structure of TmaDAH7PS with Tyr bound is presented. Tyr binding was shown to induce a significant conformational change, and Tyr is observed to bind at the interface between the ACT domains from two diagonally located monomers of the tetramer. The major reorganisation of the regulatory domain with respect to the barrel observed in the crystal structure, was confirmed by small angle X-ray scattering. The closed conformation adopted by the protein on Tyr binding physically gates the neighbouring barrel and blocks substrate entry into the active site. Chapter 5 explores the interactions between TmaDAH7PS and the allosteric inhibitor, Tyr. The residues His29 and Ser31, which form hydrogen bonds with the hydroxyl moiety of the Tyr ligand, were examined for their impact on the sensitivity and selectivity of the enzyme for the inhibitors Tyr and Phe. The hydroxyl side chain of Ser31 was found to be important for both the preferential inhibition by Tyr over Phe and the inhibitory mechanism. His29 (the hydrogen-bonding partner of Ser31) appears to play a secondary role in determining ligand selectivity and the relative positioning of these two residues is crucial to the inhibition of the enzyme. Chapter 6 evaluates the transferability of allosteric control of catalytic activity. The ACT domain of TmaDAH7PS was fused onto the barrel of the unregulated PfuDAH7PS. This chimeric enzyme was found to be catalytically active, inhibited by Tyr (although less sensitive) and preliminary crystallographic results show inhibition occurs via the same conformational change observed for wild-type TmaDAH7PS.

Dysfunction of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to the cognitive deficits of many neurodegenerative diseases and psychiatric disorders. Cognitive symptoms of Alzheimer's disease can be treated with NMDA receptor antagonists or drugs targeting the cholinergic system; however, there are no effective treatments for cognitive deficits of schizophrenia or Huntington's disease. With the discovery of a potent and selective allosteric modulator of the NMDA receptor, there is the possibility of new treatments based on NMDA receptor functional-enhancement through neuroactive steroids, closely related in structure to the endogenous neurosteroid, cerebrosterol. The aim of this thesis was to examine steroidal modulation of the NMDA receptor both in vitro and in vivo. In chapter 2, NMDA receptor enhancement of both the synthetic and endogenous neuroactive steroids was assessed in neurons maintained in cell culture using calcium imaging techniques. Sulphation of the steroids greatly increased the efficacy of NMDA receptor enhancement compared to the unsulphated steroids. Chapters 3 and 4 investigate the potential for neuroactive steroids to treat cognitive impairments of Huntington's disease. Using a mouse model, tests were selected that were analogous to those in which patients are impaired; however, no impairments were found in the mouse model. Chapter 5, therefore, used a different model of cognitive impairment – namely, rats with a set-shifting impairment, as is seen in many psychiatric and neurological disorders, including Huntington's disease – to assess the effect of the synthetic steroid administration. Unfortunately, the rats did not show the expected impairment. The lack of reliable animal models compromised testing the efficacy of these promising NMDA receptor positive allosteric modulators. Nevertheless, the promising in vitro results suggest that there could still be therapeutic potential. In addition, the compound is a useful research tool for exploring NMDA receptor function in health and disease.

Dans l’étude des GPCRs, il a longtemps été considéré que ceux-ci étaient présents à la membrane sous forme de monomères et que chaque monomère pouvait être activé par un ligand et transmettre le signal grâce à une protéine G. Certains résultats comme des courbes de compétition ou d’activation à deux phases étaient difficilement explicables avec ce modèle 1 ligand, 1 récepteur, 1 protéine G.<p>Ces dernières années, il a été montré via différentes techniques que les GPCRs étaient présents à la membrane non pas sous forme de monomères mais plutôt de dimères, de rangées de dimères voire de multimères. Cette disposition permet plus facilement d’expliquer certains résultats mais complique considérablement le modèle. Un nombre important de nouvelles questions sont posées quant au fonctionnement d’une telle formation de récepteurs. Quelle est l’unité fonctionnelle ?Y a-t-il une communication entre les récepteurs qui forment ces oligomères ?Est-ce qu’un ligand peut activer plusieurs récepteurs, plusieurs protéines G ?Que se passe-t-il quand l’oligomérisation se fait entre des récepteurs différents ?Est-ce qu’un ligand d’un récepteur peut en activer un autre ?Est-ce que la voie d’activation change en fonction du ligand ou du nombre de molécules liées sur un homomère ou un hétéromère? Toutes ces questions sont à prendre en considération lors de l’étude pharmacologique de n’importe quelle drogue car si le récepteur cible hétéromérise avec un autre, la drogue étudiée ne pourrait plus avoir le même effet. Prendre en considération tous ces paramètres peut compliquer considérablement l’étude d’une drogue mais elle pourrait en prévoir précisément certains effets secondaires.<p>Dans cette thèse nous étudions la question de la communication par la coopérativité négative (effet négatif sur l’affinité d’un site de liaison lors de la liaison du ligand sur un l’autre site du dimère). Nous mettons en évidence un lien entre l’activité constitutive d’un récepteur et le niveau de coopérativité négative grâce à des expériences d’accélération de dissociation de ligand traceur. La coopérativité négative observée sur les mutants du récepteur à la TSH les plus constitutifs est quasiment nulle. Nous montrons que cette perte de coopérativité négative provoque un changement de stœchiométrie ligand/récepteur et qu’au lieu de lier un seul ligand par dimère, les mutants les plus constitutifs sont capables d’en lier deux.<p>Malgré nos efforts pour identifier le ou les domaines de communication entre les récepteurs nous n’avons pu les déterminer. C’est pourquoi nous laissons cette question ouverte mais proposons sur base de nos observations et de la littérature, un modèle où la communication entre récepteurs pourrait être expliquée par la liaison de la protéine G au dimère de récepteur.<p> / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena. In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d. Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research. This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.