Assay technologies for GPCRs and their associated G-proteins are in demand for drug screening and other biotechnology applications such as biosensors for diagnostic purposes or odorant/flavour assessment as well as for elucidating the remaining controversial mechanisms in G-protein mediated signalling. This study aims to make progress towards developing a TR-FRET assay for G-protein interactions that could be used as a generic assay platform for GPCR signalling that would be fluorescent, homogeneous and amenable to miniaturization. The first chapter of this study investigates the use of small molecule labels, CS124-DTPA-EMCH:Tb and Alexa546 in a TR-FRET assay. This TR-FRET pair had previously been applied to Gα, Gβγ and RGS4 proteins and during the characterization of this assay, the protein CrV2 was observed to interact with the G-protein. Using TR-FRET, it was demonstrated that a high affinity interaction appears to occur between Gαi1 and CrV2 (Apparent Kd 6.2 nM). CrV2 is encoded by a polydnavirus from endoparasitoid wasps, which is thought to mediate immune suppression, and the interaction with Gα could have important implications in the regulation of the immune system of invertebrates. Improvements to the labelling strategy used in this assay are then attempted through the creation of various G-protein subunit fusions with small, genetically encoded lanthanide binding tags (LBTs) or tetracysteine motifs (TCMs) for site-specific labelling with terbium or FlAsH, respectively. The consequence of the fusions on maintaining G-protein subunit integrity and on the affinity of the tags for their labels is characterized, and then the utility of these constructs as TR-FRET partners is demonstrated. TCM:FlAsH complexes could successfully be used as TR-FRET acceptors for CS124-DTPA-EMCH:Tb labelled binding partners. The interaction between Gβγ2-TCM:FlAsH and Gα:Tb could be measured using TR-FRET and generated an apparent Kd of 3.6 nM. Likewise, LBT:Tb complexes could be used as TR-FRET donors to Alexa546 labelled binding partners which was demonstrated using the chimeric, promiscuous Gα subunit, LBT2:Tb-GαS25 and Gβγ:Alexa. Furthermore, the two site-specific labelling strategies can be used together in TR-FRET studies and an interaction between LBT2:Tb-Gα[subscript]S25 and Gβγ₂-TCM:FlAsH was shown to have an apparent Kd of 2.3 nM. The TRFRET assays were further validated using protease treatments and the addition of unlabelled binding partners reduced the TR-FRET signal. Finally, the feasibility of fusing lanthanide binding tags to GPCRs for alternate assay platforms or other applications was investigated. The β₂- adrenergic and M₂-muscarinic receptors were fused to LBTs and the integrity of the receptors determined using ligand binding and [[superscript]35 S]GTPγS signalling assays. Terbium binding to the LBT was then demonstrated. The utility of these constructs in alternative TR-FRET platforms with Gproteins was then explored. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363937 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Bien que la régulation des canaux calciques par les récepteurs couplés aux protéines G soit connue depuis une trentaine d'année, ce n'est que récemment qu'il a été découvert que ce phénomène pouvait passer par une interaction directe entre ces deux partenaires. Les RCPGs sont les senseurs d'un grand nombre de paramètres (des simples photons aux molécules odorantes en passant par des hormones, acides aminés et nucléotides) et ils contrôlent un grand nombre de processus cellulaires en fonction de ces différents stimuli, ce qui en fait une cible thérapeutique majeure. Une de leurs cibles est l'activité des canaux calciques voltage dépendants qui est responsable d'un grand nombre de processus tels que le contrôle du potentiel de membrane, le relargage de neurotransmetteurs, la contraction musculaire ou, bien sûr, le contrôle du taux de calcium intracellulaire qui est lui-même un second messager impliqué dans de nombreuses voies de régulations.Il nous a donc paru intéressant de se pencher plus en avant sur ces interactions et de trouver une méthode nous permettant de cribler ces interactions potentielles avec des RCPG ciblés pouvant intervenir dans une thématique de contrôle de la douleur. Pour cela nous avons développé une stratégie de FRET en temps résolu utilisant les cryptates de terres rares à déactivation lente couplés aux ligands du tag SNAP comme donneurs de fluorescence, les canaux calciques étudiées étant fusionnés avec cet épitope et l'eGFP fusionnée aux RCPGs en tant qu'accepteur. Ce test nous a permis de confirmer l'interaction entre CaV2.2 et ORL1 le récepteur de la nociceptine. Nous avons ensuite cherché à caractériser plus précisément cette interaction et nous avons déterminé quelles en étaient les séquences peptidiques responsables au sein des domaines C-terminaux de ces deux protéines grâce à des expériences de GST-pull down. Nous avons synthétisé un peptide reproduisant la séquence d'interaction d'ORL1 que nous avons couplé à la séquence TAT, le rendant ainsi capable de pénétrer les membranes cellulaires. Lorsque nous ajoutons ce peptide leurre dans les expériences de TR-FRET, l'augmentation de fluorescence observée en présence de CaV2.2-SNAP et ORL1-GFP disparait totalement alors que l'ajout d'un peptide contrôle composé des mêmes acides aminés mais présentés dans le désordre n'a aucun effet. Nous avons ensuite cherché à étudier les effets de ce peptide in vivo lors d'un protocole de tolérance à la morphine étant donné que les souris K.O. pour le gène d'ORL1 sont résistantes à l'apparition de cette tolérance. Cette stratégie de découplage CaV2.2 :: ORL1 abolit complètement le phénomène de tolérance aux effets analgésiques de la morphine par une action au niveau spinal. Ce travail peut conduire à l'utilisation d'une telle approche dans une perspective thérapeutique visant à améliorer l'utilisation de morphiniques lors du traitement des douleurs chroniques. / The regulation of the calcium channels by GPCRs has been known for almost thirty years but the direction interaction between those two proteins is a recent breakthrough. GPCRs are sensors for a great number of parameters (photons, smell molecules, hormones, amino acids, nucleotides…) and they control numerous cellular functions according to those parameters making them a major target for pharmacology. One of the GPCR's targets is the calcium channel activity which is responsible for a great number of cellular processes like control of the membrane potential, neurotransmitters or hormonal secretion, muscular contraction and, of course, control of the intracellular calcium level which is a second messenger of numerous cell-regulation pathways.It appears to us that it would be interesting to study more closely those interactions and find a way to screen the GPCR/calcium channels interactions that may occur in pain regulation. We developed a strategy of time resolved FRET, using rare earth cryptate coupled to the ligand of the SNAP tag which is fused to the calcium channel as fluorescence donor and eGFP fused GRPRs as acceptors. That test confirmed the interaction between CaV2.2 and ORL1, the nociceptin receptor. We characterized more precisely the peptide sequence of the carboxy-terminal domain of the two proteins which is responsible for the interaction using GST-pull down experiments. We synthesized a peptide reproducing the ORL1 interaction sequence coupled to the TAT sequence allowing to go through the cell membranes. When we add this decoy peptide to ours TR-FRET experiments we lose all the increase of fluorescence that we see in presence of CaV2.2-SNAP and ORL1-GFP but the adding of a control peptide made of the same peptides but scrambled didn't affect the experiment. Then we look for the effects of this peptide in vivo, during a morphine tolerance protocol as it was reported that the ORL1 knock-out mice were insensitive to this phenomenon. This strategy of uncoupling CaV2.2 and ORL1 leads to a complete suppression of the tolerance to the analgesic effects of the morphine by an action at the spinal level. This work could lead to a therapeutic use of this approach which could enhance the use of morphinic compounds in treatment of chronics pains.

Devant l’incidence de la suractivation de la voie PI3K/Akt/mTOR sur les cancers, nous avons choisi d’inhiber cette voie de signalisation. Etant donné la forte analogie structurale qui existe entre les enzymes PI3K et mTOR, nous avons conçu des inhibiteurs doubles ciblant deux kinases majeures de la voie. Ces inhibiteurs possèdent un noyau original pyrido[3,2-d]pyrimidinique. Afin d’apporter de la diversité fonctionnelle et d’engendrer un effet thérapeutique, les sommets C-4, C-2 et C-7 furent fonctionnalisé séquentiellement selon l’ordre suivant. Tout d’abord, la position C-4 fut fonctionnalisée par des hétérocycles par substitution nucléophile aromatique. Puis divers cycles hétéroaromatiques furent introduits en position C-2 par couplage de type Suzuki-Miyaura. Enfin, des groupements variés furent insérés en position C-7 par différentes réactions. Dans l’étude de l’influence du squelette, l’isomère de position pyrido[2,3-d]pyrimidine fut également synthétisé et fonctionnalisé. Afin de tester ces inhibiteurs originaux, une plateforme de tests d’activités in vitro a été mise en place où cinq kits enzymatiques ont été optimisés sur la kinase PI3K, et un test sur mTOR. Ces tests exploitant la méthode de TR-FRET et de bioluminescence ont été validés avec des inhibiteurs de référence basés sur 4 facteurs : la corrélation entre IC50(littérature) et IC50(mesurée), Z’, R², et S/B.Au final, plus de soixante produits finaux ont été évalués in vitro sur PI3K et mTOR. La moitié présentent une IC50 inférieure à 100 nM et 5 ont des IC50 inférieures à 10 nM. Dans le cadre des échanges du Cancéropôle Grand Ouest, les produits ont été testés sur 6 lignées de cellules cancéreuses. / Considering the impact of overactivation of the PI3K/Akt/mTOR pathway in cancer, we chose to inhibit this signaling pathway. Given the high structural similarity between the PI3K and mTOR enzymes, we designed dual inhibitors targeting two of the three major kinases of the pathway. These inhibitors posses an original pyrido[3,2-d] pyrimidine scaffold. In order to provide a functional diversity and generate a therapeutic effect, the peaks C-4, C-2 and C-7 were functionalized sequentially in the following order. Position C-4 was first functionalized with aliphatic heterocycles by nucleophilic aromatic substitution. Then, various heteroaromatic rings were introduced at the C-2 position by Suzuki-Miyaura coupling. Finally, different functions were inserted at the C-7 peak by different reactions. In order to study the influence of the scaffold, the pyrido[2,3-d]pyrimidine isomer was also synthesized and functionalized. To test these original inhibitors a platform testing in vitro activities was set up in which five assay kits were optimized for the kinase PI3K, and one kit for mTOR. These tests exploit the TR-FRET and bioluminescence methods and were validated with commercially available inhibitors based on four factors: the correlation between IC50(literature) and IC50(measured), Z’, R², and S/B. In the end, more than sixty final products were evaluated in vitro on PI3K and mTOR. Half present an IC50 below 100 nM and 5 of them show an IC50 under 10 nM. As part of collaboration within the Cancéropôle Grand Ouest, the products were also tested on six cancer cell lines.

Les récepteurs de l’ocytocine (OTR) et de la vasopressine (AVPR) sont connus pour être impliqués dans la modulation d’effets centraux complexes. Récemment l’OTR a été proposé comme une cible thérapeutique pour le traitement des troubles du spectre autistique (TSA).Afin de mieux comprendre le rôle de l’OTR et des AVPR dans les TSA, d’éclaircir des nouveaux traits de sa pharmacologie et d’établir des méthodes du criblage sur les récepteurs sauvages, nous avons développé des traceurs pour la tomographie par émission des positons ainsi que des sondes fluorescentes pour la famille OT/AVP des RCPG. Les ligands fluorescents ont été utilisés pour établir un test de liaison TR-FRET pour l’OTR et pour initier le développement du test alternatif sur les récepteurs sauvages. Les radiotraceurs TEP seront bientôt testés chez la souris et chez le singe pour évaluer leurs performances pour la détection des récepteurs de l’ocytocine centraux avant d’envisager des études chez l’Homme. / In order to better understand the role of OTR and AVPR in ASD, to reveal new features in its pharmacology and signaling and to establish high-throughput screening method on wild-type G protein-coupled receptors, we developed imaging probes for the oxytocin-vasopressin receptors family, namely radiotracers for positron emission tomography and optical probes for fluorescence detection and imaging. The fluorescent ligands have been used to establish TR-FRET binding assay for OTR and to initiate the development the screening assay for the wild-type oxytocin receptor. The PET radiotracers will be shortly tested in mice and monkeys to evaluate their potency in detecting the central oxytocin receptors.

Les récepteurs couplés aux protéines G constituent la plus grande famille de protéines membranaires et interviennent dans de nombreux processus physiologiques. La compréhension de l’interaction ligand-récepteur d’un point de vue mécanistique mais également thérapeutique est cruciale. Appartenant à la famille des récepteurs couplés aux protéines G, les récepteurs de la vasopressine et de l’ocytocine ont été choisis comme modèle d’étude. Ces hormones jouent un rôle important dans la modulation de l’attachement et de l’affect chez les mammifères. Afin d’accélérer la découverte de ligands ocytocinergiques et d’explorer les mécanismes fondamentaux de leurs interactions, nous avons conçu les premiers ligands fluorescents non peptidiques des récepteurs de la vasopressine V1a et de l’ocytocine. Ces ligands ont été utilisés pour développer des tests de liaisons par TR-FRET et démontrer la dimérisation des récepteurs de la vasopressine V1a et V2 sur cellules. Des études autour de petites plates-formes dérivées d’aza-dicétopipérazine ont permis d’accéder à un nouvel antagoniste non peptidique du récepteur de l’ocytocine. L’optimisation de dérivés benzodiazépines ocytocinergiques par des études de relations structure-activité a permis d’identifier les meilleurs agonistes non peptidiques du récepteur de l’ocytocine à ce jour. Une étude in vivo chez la souris et chez le singe est amorcée pour apporter dans un futur, une solution thérapeutique aux problèmes d’interaction sociale en général et d’autisme en particulier. / G protein coupled receptors are the largest membrane protein family and play an important role in a large number ofphysiological processes. The comprehension of the ligand-receptor interaction from a mechanistic point of view but alsofor therapeutic use is crucial. Belonging to the G protein coupled receptors, the oxytocin and vasopressin receptors havebeen used as a model system. These two hormones play an important role in the modulation of attachment and affectin mammals. To accelerate the discovery of new ligands for oxytocin and vasopressin receptors and to explore thefundamental role of their interactions, we designed the first non-peptide fluorescent ligands for oxytocin and vasopressin V1a receptors. These ligands have been used to develop new binding tests based on TR-FRET technology and to prove the V1a and V2 receptor dimerisation. In parallel, we developed a new non-peptide oxytocin antagonist around an aza-diketopiperazine platform. . Optimization of benzodiazepine derivatives enables us to identify the best non peptideoxytocin agonists to date. In vivo studies in mice and monkeys are initiated to bring in the future a therapeuticsolution to social interaction problems in general and autism in particular

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

No description available.