No description available.

No description available.

No description available.

No description available.

<p>Allosteric modulators are a newly emerging concept in the field of drug discovery which have shown remarkable success in their ability to alter G-protein coupled receptor (GPCR) activity in a precise and subtle manner. A GPCR of particular interest for allosteric targeting is the dopamine D2 receptor. This receptor has repeatedly been implicated in the etiology of complex neurological and neuropsychiatric disorders including Parkinson’s disease and schizophrenia. Previous studies from our lab have effectively developed allosteric modulators targeting the D2 receptor based on the pharmacophore of the endogenous tripeptide L-prolyl-L-leucyl-glycinamide (PLG). PLG and its potent peptidomimetics, particularly 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) (PCT/CA2011/000968), have shown robust preclinical efficacy in treating models of Parkinson’s disease, depression, tardive dyskinesia and schizophrenia. These ligands modulate agonist binding to the D2 receptor in a biphasic manner, although further information on their mechanisms of action are currently unknown. Therefore, the overarching objective of this thesis was to enhance our knowledge on the mechanisms of action of the promising D2 allosteric ligands PLG and PAOPA. Results of the studies presented here show PAOPA to cause significant upregulation of D2 regulatory proteins and downstream signaling kinases, as well as cause an increase in D2 internalization. Additionally, the PLG allosteric binding site was narrowed down to be localized between transmembrane domains 5 and 6 on the D2 receptor. The collection of work presented here enhance our understanding of the mechanisms of action of the potentially therapeutic D2 allosteric ligands PLG and PAOPA, progressing them closer to helping clinically affected populations. The findings of these studies prove globally significant as they highlight the diverse cellular pathways which could be affected by allosteric modulators, and bring to light the importance of studying these candidate ligands for eventual improvements in the treatment of human health.</p> / Doctor of Philosophy (Medical Science)

Schizophrenia is a severe mental disorder that can manifest in various ways and is often characterized by the appearance of positive symptoms (hallucinations, delusions), negative symptoms (social and attention impairment) and cognitive dysfunction (thought disorders, memory and executive function impairments). Traditional treatment methodologies involve blocking the dopamine receptor by binding to the same site as dopamine. These treatments are largely inadequate and lead to an array of adverse side effects. Side effects include weight gain, diabetes, and movement disorders; which critically limit the therapeutic value of antipsychotic drug treatment. Limited symptom control and severe adverse effects have led to poor drug adherence and a deprived quality of life for patients suffering from schizophrenia. The complex etiology of schizophrenia combined with a lack of effective translational models and tests to represent and assess the illness have hindered drug development. Evidently, there is a strong demand for a new generation of pharmaceuticals and an improved translational pipeline for the treatment of schizophrenia. The collection of studies presented here contribute to the advancement of translational tools for drug discovery, the establishment of pre-clinical models to embody the various symptoms, and the development of a novel drug candidate for schizophrenia. Allosteric modulation of G-protein coupled receptors is evolving as a new wave of therapy with promising implications for various CNS disorders. Allosteric compounds regulate binding without blocking the receptor. PAOPA, a dopamine D2 receptor allosteric modulator, prevents and treats schizophrenia-like symptoms in pre-clinical animal models of schizophrenia with no apparent adverse effects. The studies outlined in this thesis further categorize PAOPA as a novel therapeutic candidate for schizophrenia. Moreover, the findings presented here provide further insight into the potential therapeutic mechanism of action of PAOPA and set the foundation for the development of a new generation of antipsychotic drugs. These studies constitute an innovative approach to expanding research in the field of drug development for schizophrenia. / Thesis / Doctor of Philosophy (PhD)

Due in large part to the growth and development of reliable surface characterization techniques, as well as to advances in the physical and chemical techniques used to modify surfaces, the technology of surface modification has seen rapid expansion over the past two decades. A major thrust of this research is the growth of controlled/"living" polymeric brushes from the surface of various substrates, an advance that promises to be a facile and reproducible way of altering surface properties. A unique initiator bearing ATRP (atom transfer radical polymerization), cleavage, and condensation functionalities was prepared and attached to the hydrolyzed surface of silica gel. Preliminary results indicate that control of reversibly terminated grafts of varying degrees of polymerization with polydispersity indices approaching 1.5 can be readily achieved-significant findings in the quest to design desired surface characteristics. Important physical characteristics may also be altered by way of varying molecular topologies. In the second major research thrust, the use of self-assembly to construct such topologies in the form of pseudorotaxanes fashioned from diverse macrocycles with multifarious guest ions is discussed. While the underlying goal was to investigate and understand the mode of complexation based on such environmental factors as substituent affects and neighboring group influences, new insight was gained on the synthetic manipulation of cooperative events-events that freely occur in nature. The complexation behavior of several functionalized bis-(meta-phenylene)-32-crown-10 macrocycles with various paraquat guest moieties was. As expected, studies indicated that electron-donating substituents on the crown ether drive association, a likely result of increased p -p interactions among host and guest species. The association between a bicyclic macrocycle and dimethyl paraquat was also investigated. Not surprisingly, binding of paraquat by the bicyclic was much stronger than the binding found in analogous macrocycles. Lastly, the endgroup functionalization of poly(propyleneimine) dendrimers with two crown ether macrocycles was performed and the complexation with host-specific guests studied. Curiously, two extreme binding regimes were found: the larger 32-membered crown ether assembly displayed anti-cooperative behavior upon complexation with paraquat, while the smaller 24-membered macrocyclic system exhibited cooperative effects with 2o ammonium ions. These cooperative results are among the very first described for non-biological systems and hint at their potential use in developing highly efficient, synthetically designed supramolecular systems. / Master of Science

Proteins, as pivotal players in biological processes, undergo evolutionary changes due to mutations, whether spontaneous or induced by external factors. These mutations lead to significant genomic differences, contributing to the emergence of new species. From the basic principles of evolution, including variation, selection, fitness, inheritance, and reproduction, to the detailed analysis of specific proteins in different taxonomic groups, this dissertation explores the intricate field of protein evolution. In this thesis the study of bacterial and eukaryotic proteins is covered. It includes the study of enzymes in bacteria, such as CCA-adding enzyme and poly(A) polymerase, providing insights into the evolutionary divergence of these vital proteins. An analysis of existing species protein sequences and the prediction of corresponding ancestral sequences reveals a putative ancestral gammaproteobacterial CCA-adding enzyme, which is functional, thermotolerant and has a high specificity for CCA incorporation and substrate interactions. To address the challenges of suboptimal protein sequence data quality, the develop- ment of the ExceS-A split aligner is presented, which provides an automated solution to search for high quality protein sequences across diverse species groups. It is designed for exon-by-exon comparisons of coding sequences. The computation of exon/intron structure, inherent in spliced alignment procedures, is crucial for distinguishing paralo- gous members within gene families. The simplicity and effectiveness of this blat-based approach offer distinct advantages, especially for genes with extensive introns and applications involving fragmented genome assemblies, outperforming established tools in these scenarios. The application of the tool ExceS-A is then demonstrated in the study of neu- ropeptide Y/RFamide-like receptors in nematodes, shedding light on the evolutionary dynamics within this G protein-coupled receptor (GPCR) family. The Neuropeptide Y/RFamide-like receptors play crucial roles in locomotion, feeding, and reproduction. This extensively studied receptor group in Caenorhabditis elegans, comprising 41 recep- tors, served as a starting point for understanding the family’s expansion in nematodes. 159 nematode genomes revealed a total of 1557 neuropeptide Y/RFamide-like receptor sequences. The high conservation of these receptors across nematoda underscores their significance while highlighting family diversification in nematode evolution, with clade-specific duplications and losses across the phylum and unique patterns observed in the genus Caenorhabditis. Further, the dissertation focuses on the detailed analysis of GPCRs, with a particular interest in the ADRB2 and ADRB1 and Y1R and Y2R receptor, unraveling their conservation patterns and investigating their roles in G protein coupling. The investigation extends to the broader context of GPCR signaling pathways, emphasizing the crucial long-distance signaling and proposing hypotheses regarding amino acid conservation within chordates. Molecular dynamics simulations are used to uncover allosteric mechanisms and networks, providing valuable insights into protein dynamics and interactions. The investigation aimed at determining whether the conservation of amino acids within the chordate group is higher along the transmission pathway of GPCRs compared to the normal shortest path. Contrary to the hypothesis, results for ADRB2 and Y2R receptors, both with ligands and G-proteins, showed no significant difference in conservation rates between weighted and unweighted paths. Analysis revealed that unweighted paths favor hydrophobic interactions, while weighted paths predominantly involve peptide bonds, emphasizing their importance in allosteric signal transmission. Possible reasons for the lack of a significant increase in conservation values include the overall high conservation of amino acids in transmembrane helix 2-6 and the need for more precise information about mutual information in conservation score calculations. Future efforts will explore modified k-shortest path algorithms to identify alternative geometrically related contacts. The dissertation concludes by highlighting the crucial role of bioinformatics in performing complex analyses and processing large datasets. The basics laid here provide a foundation for interdisciplinary collaboration and contribute significant insights into the evolution of proteins. As a comprehensive knowledge framework, this work is able to guide future research efforts and underscores the ongoing importance of uncovering the complex interactions that govern protein evolution in the field of biological research.

L’approche Module X a été créée dans le but de concevoir de petits peptides modulateurs ayant des propriétés allostériques. Module X reproduit de petites parties des portions extracellulaires flexibles des récepteurs. Ces petits peptides vont interagir en s’interposant entre deux sous unités ou entre deux régions de la même sous-unité qui interagissent par des liens hydrogènes, des ponts salins ou des liens disulfure. Ces régions sont spécialement choisies à l’extérieur du domaine de liaison du ligand orthostérique et sont situées dans les régions inter domaines, la portion juxta membranaire ou dans les boucles. Étant donné que les boucles sont exposées durant les changements de conformation, une séquence peptidique reproduisant certaines régions de ces boucles pourrait s’insérer à un endroit approprié dans la structure où se lier à son partenaire de signalisation dans le complexe protéique, ce qui aurait comme effet de déplacer l’équilibre de l’ensemble vers un état particulier et modulerait ainsi la signalisation. De cette façon, certaines voies de signalisation pourraient être partiellement inhibées tandis que d’autres voies ne seraient pas touchées puisque le ligand orthostérique pourrait toujours se lier au récepteur. Dans une première étude, nous avons conçu des peptides inhibiteurs du récepteur de l’interleukine 1 (IL-1R/IL-1RAcP) plus précisément en reproduisant des régions flexibles de la protéine accessoire, sous-unité signalisatrice du récepteur. IL-1 est un médiateur majeur de l’inflammation, mais le seul antagoniste disponible est l’analogue naturel de IL-1, IL-1Ra qui compétitionne avec IL-1 pour le site de liaison sur le récepteur. Nous avons conçu plusieurs peptides à partir des boucles de la protéine accessoire. Un de ces peptides, rytvela (101.10) a démontré des propriétés de non-compétitivité et de sélectivité fonctionnelle caractéristiques des modulateurs allostériques. 101.10 bloque la prolifération des thymocytes et la synthèse de PGE2 avec un IC50 de 1 nM mais une efficacité de 100 % et 45 % respectivement et ne déplace pas IL-1 radioactif dans des essais de radioliaisons. De plus, 101.10 n’a qu’un effet minime sur l’affinité de IL-1 pour son récepteur. 101.10 démontre, de plus, une activité inhibitrice in vivo dans des modèles d’inflammation de l’intestin chez le rat (efficacité supérieure aux corticostéroïdes et à IL-1Ra) et de dermatite chez la souris de même que dans un modèle d’hyperthermie induite par IL-1. La deuxième étude démontre que Module X peut être utilisé pour concevoir des inhibiteurs pour une autre grande famille de récepteurs : les récepteurs couplés aux protéines G. La vasopressine joue un rôle important dans l’équilibre hydro-osmotique et un moindre rôle dans la vasomotricité. Six peptides ont été conçus à partir de régions juxta membranaires du récepteur de la vasopressine V2R. Le peptide le plus actif, VRQ397 (IC50 = 0,69 nM dans un modèle de vasorelaxation du crémastère), a démontré de la sélectivité fonctionnelle en inhibant la synthèse de prostacycline mais sans inhiber l’activation de la protéine Gs et la génération d’ AMP cyclique. Le peptide VRQ397 ne pouvait déplacer le ligand naturel AVP marqué radioactivement; de même VRQ397 radioactif ne se liait que sur V2R et non pas sur d’autres récepteurs de la même famille tel que V1R (récepteur de la vasopressine de type I). Ces études décrivent la caractérisation de petits peptides modulateurs de la signalisation de IL-1R et V2R et présentant des propriétés de modulateurs allostériques. / The Module X approach was conceived to generate small allosteric peptides that do not (by definition) compete with the natural ligand to inhibit or modulate signalling. Orthosteric inhibition blocks the entire signalling pathways while allosteric modulators will bind to another site on the target and show functional selectivity. By reproducing parts of the flexible regions (loops) of two receptors, the IL-1 and vasopressin receptors, we generated small peptides that showed allosteric properties. To prove our concept we started with a pro-inflammatory target: IL-1 receptor. Interleukin (IL)-1 is a major pro-inflammatory cytokine which interacts with the IL-1 receptor I (IL-1RI) complex, composed of IL-1RI and IL-1R accessory protein (IL-1RacP) subunits. Presently, there are no small antagonists of the IL-1RI complex. Given this void, we derived 15 peptides from loops of IL-1RacP, which are putative interactive sites with the IL-1RI subunit. Here we substantiate the merits of one of these peptides, rytvela (we termed, 101.10), as an inhibitor of IL-1R and describe its properties consistent with those of an allosteric negative modulator. 101.10 (IC50  1 nM) blocked human thymocyte proliferation in vitro, and demonstrated robust in vivo effects in models of hyperthermia and inflammatory bowel disease as well as topically in contact dermatitis, superior to corticosteroids and IL-1ra; 101.10 did not bind to IL-1RI deficient cells and was ineffective in vivo in IL-1RI knockout mice. Importantly, characterization of 101.10, revealed non-competitive antagonist actions and functional selectivity by blocking certain IL-1R pathways while not affecting others. The second study involved a representative of the biggest family of membrane proteins: G-protein coupled receptors. Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydro-osmotic equilibrium and to a lesser extent on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, vasopressin (AVP), and hence are competitive ligands. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, CRAVKY (labelled VRQ397), was characterized. VRQ397 was potent (IC50 = 0.69 ± 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function (specifically DDAVP-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events). Dose-response profile revealed non-competitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells, could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). VRQ397 exhibited pharmacological permissiveness on V2R-induced signals as it inhibited DDAVP-induced PGI2 generation, but not that of cAMP or recruitment of — arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. Findings describe the discovery of potent and specific small (peptide) antagonists of IL-1RI and V2R with properties in line with an allosteric negative modulator.

Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound. In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes. An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness. Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products.