Alzheimers disease (AD) represents one of the greatest health problems in industrialized countries considering the ageing population. Only four drugs are currently approved for the treatment of this disease. As these drugs are characterized with a limited time efficacy, it has become urgent to develop additional innovative AD treatments. Amongst the approaches that are actively investigated, the one consisting in potentiating a subclass of glutamate receptors appears attractive. This well advanced pharmacological approach includes three major classes of compounds amongst which appear the benzothiadiazine 1,1-dioxides. The present thesis is a pursuit of the preliminary efforts that were published in 1998 and 2001 by our team. Based on promising in vitro results obtained with the lead compound 59, pharmacomodulations around 59s structure have been achieved in order to enhance its in vivo activity and to optimize its pharmacokinetic parameters. First efforts were devoted to exploratory synthesis where attention was paid to the impact of the substituent introduced at the 7-position. Moreover, some pyridothiadiazine dioxides as well as thienothiadiazine dioxides were prepared. The most important part of our pharmacomodulations was focused on the thiadiazine ring system. Considering that the poor in vivo results obtained with 59 could be due to a metabolic weakness of the latter, the introduction of fluorine atoms was tempted as a lead optimization strategy. This approach was successful, since it led to the synthesis of 95b which was selected for further pharmacological evaluations. This new lead compound was shown to exert significant cognitive-enhancing effects in vivo after oral administration to Wistar rats. Moreover, the study of the metabolic degradation of 95b allowed the assessment of the starting hypothesis that had dictated the pharmacomodulations philosophy. Finally, additional exploratory pharmacomodulations were achieved notably leading to the preparation of a quinazolinone series and 1,4-benzothiazine compounds. This research allowed to significantly improve the pharmacokinetic profile of our series and led to the identification of 95b as a new lead compound. However, many pharmacomodulations remain to be explored. The data collected during this thesis are appealing further studies. Efforts in the near future should lead to the design of novel drug candidates among which a future innovative AD treatment could emerge. Au vu du vieillissement de la population dans les pays industrialisés, la maladie dAlzheimer constitue un problème majeur en termes de santé publique. A lheure actuelle, seuls quatre médicaments sont utilisés pour traiter cette maladie. Sachant que ces substances nont quune efficacité limitée, la conception de nouveaux médicaments actifs contre cette pathologie apparaît comme une priorité. Dans cette optique, la potentialisation des récepteurs glutamatergiques de sous-type AMPA semble une approche thérapeutique intéressante. Parmi les composés étudiés dans cette voie, apparaissent les benzothiadiazine dioxydes. Cette thèse sinscrit dans la continuité des recherches publiées en 1998 et 2001 par notre équipe et poursuit les pharmacomodulations entamées autour du composé leader 59. Ce travail sest principalement focalisé sur lamélioration de lactivité in vivo de ce dérivé et sur loptimisation de ses paramètres pharmacocinétiques afin dobtenir un candidat médicament potentiel. Notre attention sest tout dabord portée sur la subsitution aromatique en position 7. Par ailleurs, plusieurs séries pyrido- et thiénothiadiazines ont été également préparées. Ensuite, en postulant que le manque dactivité in vivo du composé 59 pouvait être dû à une faiblesse métabolique de ce composé, lintroduction judicieuse datomes de fluor a été choisie comme stratégie doptimisation. Cette approche nous a notamment amené à lidentification du composé 95b. Ce dérivé sest montré particulièrement actif dans un test de reconnaissance dobjets chez le rat Wistar. Ce résultat encourageant a amené à réaliser toute une série dévaluations pharmacologiques sur ce produit, afin de caractériser son mécanisme daction. Les données récoltées à lissue de ces investigations suggèrent que le composé 95b présente un intérêt potentiel en tant quagent procognitif. Dautre part, létude du profil de dégradation métabolique de ce dérivé a permis de confirmer lhypothèse qui avait dicté lintroduction datomes de fluor. Enfin, des pharmacomodulations supplémentaires ont été réalisées de façon exploratoire et ont entre autres débouché sur une série de quinazolinones et une série de 1,4-benzothiazines. Les travaux réalisés au cours de cette thèse ont permis daméliorer grandement le profil pharmacocinétique des séries explorées. Néanmoins, de nombreuses pharmacomodulations restent à explorer. Des études complémentaires savèrent nécessaires et devraient déboucher sur de nouveaux candidats médicaments innovants, parmi lesquels pourraient figurer un futur traitement de la maladie dAlzheimer.

abstract: G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set. The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

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No description available.

<p>The neurosteroids constitute a group of powerful hormones synthesized and acting in the central nervous system. They participate in a number of important central processes, such as memory and learning, mood and neuroprotection. Their effects emerge from rapid interactions with membrane bound receptors, such as the N-methyl-D-aspartate (NMDA) receptor, the gamma-amino-butyric acid receptor and the sigma 1 receptor. The mechanisms of action are separate from classical genomic interactions. </p><p>The aims of this thesis were to identify and characterize the molecular mechanisms underlying the effects of nanomolar concentrations of neurosteroids at the NMDA receptor. </p><p>The results show that the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate 3α5βS) differently modulate the NMDA receptor, changing the kinetics for the NMDA receptor antagonist ifenprodil, through unique and separate targets at the NR2B subunit. The effects that appear to be temperature independent were further confirmed in a calcium imagining functional assay. A second functional study demonstrated that PS and 3α5βS affect glutamate-stimulated neurite outgrowth in NG108-15 cells. </p><p>Misuse of anabolic androgenic steroids (AAS) has powerful effects on emotional states. Since neurosteroids regulate processes involved in mood it can be hypothesised that AAS can interact with the action of neurosteroids in the brain. However, chronic administration of the AAS nandrolone decanoate did not alter the allosteric effects of PS or 3α5βS at the NMDA receptor, but changed the affinity for PS, 3α5βS and dehydroepiandrosterone sulfate to the sigma 1 receptor. The results also showed that the neurosteroids displace ³H-ifenprodil from the sigma 1 and 2 receptors without directly sharing the binding site for ³H-ifenprodil at the sigma 1 receptor. The decreased affinity for the neurosteroids at the sigma 1 receptor may be involved in the depressive symptoms associated with AAS misuse.</p><p>The NMDA receptor system is deeply involved in neurodegeneration and the NMDA receptor antagonist ifenprodil exert neuroprotective actions. The findings that neurosteroids interact with ifenprodil at the NMDA receptor may be an opportunity to obtain synergistic effects in neuroprotective treatment.</p>

The neurosteroids constitute a group of powerful hormones synthesized and acting in the central nervous system. They participate in a number of important central processes, such as memory and learning, mood and neuroprotection. Their effects emerge from rapid interactions with membrane bound receptors, such as the N-methyl-D-aspartate (NMDA) receptor, the gamma-amino-butyric acid receptor and the sigma 1 receptor. The mechanisms of action are separate from classical genomic interactions. The aims of this thesis were to identify and characterize the molecular mechanisms underlying the effects of nanomolar concentrations of neurosteroids at the NMDA receptor. The results show that the neurosteroids pregnenolone sulfate (PS) and pregnanolone sulfate 3α5βS) differently modulate the NMDA receptor, changing the kinetics for the NMDA receptor antagonist ifenprodil, through unique and separate targets at the NR2B subunit. The effects that appear to be temperature independent were further confirmed in a calcium imagining functional assay. A second functional study demonstrated that PS and 3α5βS affect glutamate-stimulated neurite outgrowth in NG108-15 cells. Misuse of anabolic androgenic steroids (AAS) has powerful effects on emotional states. Since neurosteroids regulate processes involved in mood it can be hypothesised that AAS can interact with the action of neurosteroids in the brain. However, chronic administration of the AAS nandrolone decanoate did not alter the allosteric effects of PS or 3α5βS at the NMDA receptor, but changed the affinity for PS, 3α5βS and dehydroepiandrosterone sulfate to the sigma 1 receptor. The results also showed that the neurosteroids displace 3H-ifenprodil from the sigma 1 and 2 receptors without directly sharing the binding site for 3H-ifenprodil at the sigma 1 receptor. The decreased affinity for the neurosteroids at the sigma 1 receptor may be involved in the depressive symptoms associated with AAS misuse. The NMDA receptor system is deeply involved in neurodegeneration and the NMDA receptor antagonist ifenprodil exert neuroprotective actions. The findings that neurosteroids interact with ifenprodil at the NMDA receptor may be an opportunity to obtain synergistic effects in neuroprotective treatment.

The glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine. / text

The glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine. / text

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.