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Peroxisome proliferator-activated receptor γ (PPARγ) is associated with a wide range of diseases, including type 2 diabetes mellitus (T2D). Thiazolidinediones (TZDs) are agonists of PPARγ which have an insulin sensitizing effect, and are therefore used as a treatment for T2D. However, TZDs cause negative side effects in patients, such as weight gain, edema, and increased risk of bone fracture. Partial agonists could be an alternative to TZD-based drugs with fewer side effects. However, there is a lack of understanding of the types of PPARγ partial agonists and how they differ from full agonists. In silico techniques, like virtual screening, molecular docking, and pharmacophore modeling, allow us to determine and characterize markers of varying levels of agonism. An extensive search of the RCSB Protein Data Bank found 62 structures of PPARγ resolved with partial agonists. Cross-docking was performed and found that two PDB structures, 3TY0 and 5TWO, would be effective as receptor structures for virtual screening. By clustering known partial agonists by common pharmacophore features, we found several distinct groups of partial agonists. Interaction and pharmacophore models were created for each group of partial agonists. Virtual screening of FDA-approved compounds showed that the models were able to predict potential partial agonists of PPARγ. This study provides additional insight into the different binding modes of partial agonists of PPARγ and their characteristics. These models can be used to assist drug discovery efforts for intelligently designing novel therapeutics for T2D which have fewer negative side effects. / Master of Science in Life Sciences / The peroxisome proliferator-activated receptor γ (PPARγ) protein is associated with a wide range of diseases, including type 2 diabetes mellitus (T2D). Thiazolidinediones (TZDs) are compounds that activate PPARγ, and increase insulin sensitivity in patients with T2D. However, TZDs cause negative side effects in patients, such as weight gain, increased fluid retention, and increased risk of bone fracture. Partial agonists could be an alternative to TZD-based drugs with fewer side effects. However, there is a lack of understanding of the types of PPARγ partial agonists and how they differ from full agonists. Computational techniques allow us to investigate common features between known partial agonists. An extensive search of the RCSB Protein Data Bank found 62 structures of PPARγ which contained partial agonists. Each known partial agonist was docked into twelve complete PPARγ structures, and it was found that two structure models would be effective as receptor structures for virtual screening. A set of known partial agonists were grouped based on common chemical features, and three distinct groups of partial agonists were found. Binding criteria for each of these three groups were developed. A library of FDA-approved compounds was screened using the criteria for binding to identify potential novel partial agonists. Three potential novel partial agonists were found in the screening. This study provides additional insight into how different compounds activate PPARγ. These methods can be used to assist drug discovery efforts for intelligently designing novel therapeutics for T2D which have fewer negative side effects.

L’objectif de ces travaux est de découvrir et de valoriser des petites molécules organiques inhibant l’influx de l’iode dans les cellules thyroïdiennes. Ces composés présentent en effet un double intérêt : à court terme, ils peuvent être dérivés en biosondes afin de mieux caractériser les protéines impliquées dans les mécanismes de transport d’iode par génétique chimique directe. A plus long terme, ces inhibiteurs représentent des candidats-médicaments potentiels pour le traitement de pathologies thyroïdiennes et/ou pour la protection de populations exposées aux radioisotopes de l’iode. Pour chacune des deux familles d’inhibiteurs considérées, nous avons donc tout d’abord synthétisé une chimiothèque d’une centaine d’analogues ; puis ces derniers ont été évalués biologiquement afin de fournir un ensemble de relations structure-activité. Par ailleurs, la configuration absolue des centres stéréogènes nécessaire à l’activité biologique a été déterminée : dans chacun des cas, une stéréochimie particulière est responsable du pouvoir inhibiteur des composés. A partir de ces informations, une dizaine d’analogues « de seconde génération » a été synthétisée dans chaque famille, en combinant plusieurs modifications structurales contribuant à l’activité biologique. Après évaluation biologique, neuf d’entre eux possèdent des IC50 < 6 nM et des propriétés physico-chimiques satisfaisantes pour des candidats-médicaments. Enfin, dans chaque famille, une biosonde photoactivable biotinylée a été synthétisée et utilisée en photomarquage d’affinité. Plusieurs protéines marquées spécifiquement ont été repérées, qui correspondraient à des protéines-cibles de chacun des inhibiteurs et dont l’identification reste à achever. / This work was intended to discover small organic molecules acting as iodide uptake inhibitors in thyroid cells. These compounds can indeed be derivatized into biochemical probes for further characterization of proteins involved in iodide transport mechanisms. On the long term, these inhibitors also appear as attractive drug candidates for treatment of thyroid pathologies or radioprotection against iodine isotopes. A similar strategy was adopted for both of the two inhibitor families. First, we synthesized a chemical library of around 100 analogues; we measured their IC50 against iodide uptake in FRTL-5 cells to get structure-activity relationships. Absolute configuration of stereogenic centers was also investigated, and a preferential stereochemistry was found to be responsible for activity. From this basis, around twenty « second-generation » analogues were synthesized by combining fragments contributing to biological activity. Biological evaluation indicated that nine were very potent inhibitors, with IC50 < 6 nM and satisfying physicochemical properties required for drug candidates. Finally, one photoactivatable biotinylated probe was developed in each family and used for photoaffinity labeling. Several specifically labeled proteins are still under identification and constitute new potential therapeutic targets.

Les interfaces protéine-protéine sont au cœur de nombreux mécanismes physiologiques du vivant. Les caractériser au niveau moléculaire est un donc enjeu crucial pour la recherche de nouveaux candidat-médicaments. Nous proposons ici de nouvelles méthodes d’analyse des interfaces protéine-protéine à visée pharmaceutique. Notre protocole automatisé détecte les interfaces au sein des structures de la Protein Data Bank afin de définir les zones d’interactions à potentiel pharmacologique, les cavités droguables, les ligands présents à l’interface ainsi que les pharmacophores directement déduits à partir des cavités. Notre méthode permet de réaliser un état de l’art des informations disponibles autour des interfaces protéine-protéine ainsi que de prédire de nouvelles cibles potentielles pour des molécules candidats médicaments. / Protein-protein interfaces are involved in many physiological mechanisms of living cells. Their characterization at the molecular level is therefore crucial in drug discovery.We propose here new methods for the analysis protein-protein interfaces of pharmaceutical interest. Our automated protocol detects the biologicaly relevant interfaces within the Protein Data Bank structures, droguables cavities, ligands present at the interface and pharmacophores derived directly from the cavities. Our method enables a state-of- the-art of all available structural information about protein-protein interfaces and predicts potential new targets for drug candidates.

Un des buts de la recherche pharmaceutique est l'inhibition de protéines avec l'aide de petites molécules (ligands). L'une des phases clefs de ce procédé est la détermination du mode d'interaction entre un ligand et son récepteur. Cette tâche peut être entravée par l'absence de structure du complexe protéine-ligand. C'est pour répondre à ce besoin que nous présentons dans ce travail de thèse, une méthode capable de déterminer la structure de complexes protéine-ligands. Dans la méthode INPHARMA (Inter-ligands Nuclear Overhauser Effect for Pharmacophore Mapping), les inter-ligands NOEs (INPHARMA NOEs) sont utilisés pour déterminer l'orientation relative de deux ligands qui interagissent de manière compétitive avec un même récepteur. Cette nouvelle approche ouvre la voie à des applications pharmaceutiques, également au stade initial du développement, quand l'information structurale via la cristallographie par Rayons X est difficile d'accès.

Schizophrenia is a chronic psychotic illness affecting ~21 million people globally. Currently available antipsychotic agents act through a dopamine D2 receptor mechanism, and produce extrapyramidal or metabolic side effects. Hence, there is a need for novel targets and agents. The mGlu2/5-HT2A receptor heteromer has been implicated in the action of antipsychotic agents, and represents a novel and attractive therapeutic target for the treatment of schizophrenia. A long-term goal of this project is to synthesize bivalent ligands where a 5-HT2A receptor antagonist is tethered to an mGlu2 PAM via a linker. The goals of the investigation were to study the SAR of risperidone (an atypical antipsychotic agent) at 5-HT2A receptors using a “deconstruction-reconstruction-elaboration” approach to determine the minimal structural features of risperidone that contribute to its 5-HT2A receptor affinity and antagonism, and to determine where on the “minimized risperidone” structure an mGlu2 PAM can be introduced. Additional goals included studying the binding modes of various mGlu2 PAMs and identifying where on an mGlu2 PAM a risperidone “partial” structure could be introduced. Biological studies of deconstructed/elaborated analogs of risperidone suggest that the entire structure of risperidone is not necessary for 5-HT2A receptor affinity and antagonism, and that a fluoro group contributes to 5-HT2A binding. 6-Fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole that has only half the structural features of risperidone retains 5-HT2A receptor affinity and antagonist activity, and represents the “minimized risperidone” structure with the piperidine nitrogen atom representing a potential linker site for eventual construction of bivalent ligands. Molecular modeling studies at 5-HT2A receptors suggest that risperidone and its analogs have more than one binding mode. Modeling studies to evaluate binding modes of various PAMs at mGlu2 receptors, coupled with known SAR information, were used to identify a PAM (JNJ-40411813), and the pyridone nitrogen atom of JNJ-40411813 as a potential linker site. Additionally, potential synthetic routes for JNJ-40411813 were explored that might be of value in the synthesis of bivalent ligands. Based on the structural features of 6-fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole, a new pharmacophore for 5-HT2A receptor antagonists, consisting of one aromatic region, a basic protonated amine and hydrogen bond acceptors, has been proposed.

Of the seven existing classes of serotonin receptors, the 5-HT7 receptors (5-HT7Rs) are the most recently discovered. Abundance of 5-HT7 in the central nervous system is suggestive of the receptor’s role in several physiological and pathophysiological functions. Existing research has afforded a number of compounds exhibiting specific affinity to the receptor. These selective ligands can provide structural information about the receptor and can serve as the foundation for pharmacological profiling . This thesis describes the synthesis of substituted pyrimidines and pyridines for affinity to the 5-HT7 receptor. Organometallic species are the cornerstone for sev-eral of the synthetic pathways.

Signal transducer and activator of transcription 3 (STAT3) is a cancer-driving proto-oncoprotein that represents a novel target for the development of chemotherapeutics. In this study, the functional requirements to furnish a potent STAT3 inhibitor was investigated. First, a series of peptidomimetic inhibitors were rationally designed from lead parent peptides. Prepared peptidomimetics overcame the limitations normally associated with peptide agents and displayed improved activity in biophysical evaluations. Notably, lead peptidomimetic agents possessed micromolar cellular activity which was unobserved in both parent peptides. Peptidomimetic design relied on computational methods that were also employed in the design of purine based STAT3 inhibitory molecules. Docking studies with lead STAT3-SH2 domain inhibitory molecules identified key structural and chemical information required for the construction of a pharmacophore model. 2,6,9-heterotrisubstituted purines adequately fulfilled the pharmacophore model and a library of novel purine-based STAT3 inhibitory molecules was prepared utilizing Mitsunobu chemistry. Several agents from this new library displayed high affinity for the STAT3 protein and effectively disrupted the STAT3:STAT3-DNA complex. Furthermore, these agents displayed cancer-cell specific toxicity through a STAT3 dependant mechanism. While purine agents elicited cellular effects, the dose required for cellular efficacy was much higher than those observed for in vitro STAT3 dimer disruption. The diminished cellular activity could be attributed to the apparent poor cell permeability of the first generation purine library; thus, a second library of purine molecules was constructed to improve cell penetration. Unfortunately, iii 2nd generation purine inhibitors failed to disrupt phosphorylated STAT3 activity and suffered from poor cell permeability. However, a lead sulfamate agent was discovered that showed potent activity against multiple myeloma cancer cells. Investigations revealed potential kinase inhibitory activity as the source of the sulfamate purine’s biological effect. Explorations into the development of a potent STAT3 SH2 domain binder, including the creation of salicylic purine and constrained pyrimidine molecules, are ongoing. Finally, progress towards the creation of a macrocyclic purine combinatorial library has been pursued and is reported herein.

Signal transducer and activator of transcription 3 (STAT3) is a cancer-driving proto-oncoprotein that represents a novel target for the development of chemotherapeutics. In this study, the functional requirements to furnish a potent STAT3 inhibitor was investigated. First, a series of peptidomimetic inhibitors were rationally designed from lead parent peptides. Prepared peptidomimetics overcame the limitations normally associated with peptide agents and displayed improved activity in biophysical evaluations. Notably, lead peptidomimetic agents possessed micromolar cellular activity which was unobserved in both parent peptides. Peptidomimetic design relied on computational methods that were also employed in the design of purine based STAT3 inhibitory molecules. Docking studies with lead STAT3-SH2 domain inhibitory molecules identified key structural and chemical information required for the construction of a pharmacophore model. 2,6,9-heterotrisubstituted purines adequately fulfilled the pharmacophore model and a library of novel purine-based STAT3 inhibitory molecules was prepared utilizing Mitsunobu chemistry. Several agents from this new library displayed high affinity for the STAT3 protein and effectively disrupted the STAT3:STAT3-DNA complex. Furthermore, these agents displayed cancer-cell specific toxicity through a STAT3 dependant mechanism. While purine agents elicited cellular effects, the dose required for cellular efficacy was much higher than those observed for in vitro STAT3 dimer disruption. The diminished cellular activity could be attributed to the apparent poor cell permeability of the first generation purine library; thus, a second library of purine molecules was constructed to improve cell penetration. Unfortunately, iii 2nd generation purine inhibitors failed to disrupt phosphorylated STAT3 activity and suffered from poor cell permeability. However, a lead sulfamate agent was discovered that showed potent activity against multiple myeloma cancer cells. Investigations revealed potential kinase inhibitory activity as the source of the sulfamate purine’s biological effect. Explorations into the development of a potent STAT3 SH2 domain binder, including the creation of salicylic purine and constrained pyrimidine molecules, are ongoing. Finally, progress towards the creation of a macrocyclic purine combinatorial library has been pursued and is reported herein.