The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the alpha1B-adrenergic receptor (alpha1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the alpha1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.

Cutting edge: thymic crosstalk regulates delta-like 4 expression on cortical epithelial cells.

Interactions between Notch1 receptors on lymphoid progenitors and Delta-like 4 (DL4) ligands on cortical thymic epithelial cells (cTEC) are essential for T cell lineage commitment, expansion, and maturation in the thymus. Using a novel mAb against DL4, we show that DL4 levels on cTEC are very high in the fetal and neonatal thymus when thymocyte expansion is maximal but decrease dramatically in the adult when steady-state homeostasis is attained. Analysis of mutant mouse strains where thymocyte development is blocked at different stages indicates that lymphostromal interactions ("thymus crosstalk") are required for DL4 down-regulation on cTEC. Reconstitution of thymocyte development in these mutant mice further suggests that maturation of thymocytes to the CD4(+)CD8(+) stage and concomitant expansion are needed to promote DL4 down-regulation on cTEC. Collectively, our data support a model where thymic crosstalk quantitatively regulates the rate of Notch1-dependent thymopoiesis by controlling DL4 expression levels on cTEC.

PPARs: nuclear receptors for fatty acids, eicosanoids, and xenobiotics.

The peroxisome proliferator-activated receptors have enjoyed the spotlight for many reasons. These transcription factors are ligand-inducible nuclear receptors that modulate gene expression in response to a broad spectrum of compounds. The recognition that PPARs are indeed nuclear receptors for polyunsaturated fatty acids, some eicosanoids and also lipid-lowering and antidiabetic drugs, has opened many exciting avenues of research and drug discovery. Recent studies on the PPAR function have extended the role of these transcription factors beyond energy homeostasis to master gene in adipogenesis and also determinants in inflammation control. While rapid advances have been made, it is clear that we are far from a global understanding of the mechanisms and functions of PPARs.

Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acide-Sensing Ion Channels)

RESUME: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse Les canaux sodiques ASICs (Acid-Sensing Ion Channels) participent à la signalisation neuronale dans les systèmes nerveux périphérique et central. Ces canaux non voltage dépendants sont impliqués dans l'apprentissage, l'expression de la peur, la neurodégénération consécutive à une attaque cérébrale et la douleur. Les bases moléculaires sous-tendant leur activité ne sont pas encore totalement comprises. Ces canaux sont activés par une acidification du milieu extracellulaire et régulés, entre autres, par des ions tels que le Ca2+, le Zn2+ et le CI". La cristallisation de ASIC inactivé a été publiée. Le canal est un trimére de sous-unités identiques ou homologues. Chaque sous-unité a été décrite en analogie à un avant bras, un poignet et une main constituée d'un pouce, d'un doigt, d'une articulation, une boule β et une paume. Nous avons appliqué une approche bioinformatique systématique pour identifier les pH senseurs putatifs de ASICIa. Le rôle des pH senseurs putatifs a été testé par mutagénèse dirigée et des modifications chimiques combinées à une analyse fonctionnelle afin de comprendre comment les variations de ρ H ouvrent ces canaux. Les pH senseurs sont des acides aspartiques et glutamiques éparpillés sur la boucle extracellulaire suggérant que les changements de pH contrôlent l'activation et l'inactivation de ASIC en (dé)protonant ces résidus en divers endroits de la protéine. Par exemple lors de l'activation, la protonation des résidus à l'interface entre le pouce, la boule β et le doigt d'une même sous-unité induit un mouvement du pouce vers la bouie β et le doigt. De même lors de l'inactivation du canal les paumes des trois sous-unités formant une cavité se rapprochent. D'après notre approche bioinformatique, aucune histidine n'est impliquée dans la détection des variations de pH extracellulaire c'est-à-dire qu'aucune histidine ne serait un pH-senseur. Deux histidines de ASIC2a lient le Zn2+ et modifient l'affinité apparente du canal pour les protons. Une seule des deux est conservée parmi tous les ASICs, hASICIa H163. Elle forme un réseau de liaison hydrogène avec ses voisins conservés. L'étude détaillée de ce domaine, Pinterzone, montre son importance dans l'expression fonctionnelle des canaux. La perturbation de ce réseau par l'introduction d'un résidu hydrophobe (cystéine) par mutagénèse dirigée diminue l'expression du canal à la membrane plasmique. La modification des cystéines introduites par des réactifs spécifiques aux groupements sulfhydryle inhibe les canaux mutés en diminuant leur probabilité d'ouverture. Ces travaux décrivent les effets de l'acidification du milieu extracellulaire sur les canaux ASICs. ABSTRACT: Study of pH-dependent activation and inactivation of ASIC channels Benoîte BARGETON, Department of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1G05 Lausanne, Switzerland The ASIC (Acid-Sensing Ion Channels) sodium channels are involved in neuronal signaling in the central and peripheral nervous system. These non-voltage-gated channels are involved in learning, the expression of fear, neurodegeneration after ischemia and pain sensation. The molecular bases underlying their activity are not yet fully understood. ASICs are activated by extracellular acidification and regulated, eg by ions such as Ca2+, the Zn2+ and CI". The crystallization of inactivated ASIC has been published. The channel is a trimer of identical or homologous subunits. Each subunit has been described in analogy to a forearm, wrist and hand consisting of a thumb, a finger, a knuckle, a β-ball and a palm. We applied a systematic computational approach to identify putative pH sensor(s) of ASICIa. The role of putative pH sensors has been tested by site-directed mutagenesis and chemical modification combined with functional analysis in order to understand how changes in pH open these channels. The pH sensors are aspartic and glutamic acids distributed throughout the extracellular loop, suggesting that changes in pH control activation and inactivation of ASIC by protonation / deprotonation of many residues in different parts of the protein. During activation the protonation of various residues at the interface between the finger, the thumb and the β-ball induces the movement of the thumb toward the finger and the β-ball. During inactivation of the channel the palms of the three subunits forming a cavity approach each other. No histidine has been shown to be involved in extracellular pH changes detection, i.e. no histidine is a pH- sensor. Two histidines of ASIC2 bind Zn2+ and alter the apparent affinity of channel for protons. Only one of the two His is conserved among all ASICs, hASICIa H163. This residue is part of a network of hydrogen bonding with its conserved neighbors. The detailed study of this area, the interzone, shows its importance in the functional expression of ASICs. Disturbance of this network by the introduction of hydrophobic residues decreases the cell surface channel expression. Chemical modification of the introduced cysteines by thiol reactive compounds inhibits the mutated channels by a reduction of their open probability. These studies describe the effects of extracellular acidification on ASICs. RESUME GRAND PUBLIC: Etude de l'activation et de l'inactivation pH-dépendantes des canaux ASICs (Acid-Sensing Ion Channels) Benoîte BARGETON, Département de Pharmacologie et de Toxicologie, Université de Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Suisse La transmission synaptique est un processus chimique entre deux neurones impliquant des neurotransmetteurs et leurs récepteurs. Un dysfonctionnement de certains types de synapses est à l'origine de beaucoup de troubles nerveux, tels que certaine forme d'épilepsie et de l'attention. Les récepteurs des neurotransmetteurs sont de très bonnes cibles thérapeutiques dans de nombreuses neuropathologies. Les canaux ASICs sont impliqués dans la neurodégénération consécutive à une attaque cérébrale et les bloquer pourraient permettre aux patients d'avoir moins de séquelles. Les canaux ASICs sont des détecteurs de l'acidité qui apparaît lors de situations pathologiques comme l'ischémie et l'inflammation. Ces canaux sont également impliqués dans des douleurs. Cibler spécifiquement ces canaux permettrait d'avoir de nouveaux outils thérapeutiques car à l'heure actuelle l'inhibiteur de choix, l'amiloride, bloque beaucoup d'autres canaux empêchant son utilisation pour bloquer les ASICs. C'est pourquoi il faut connaître et comprendre les bases moléculaires du fonctionnement de ces récepteurs. Les ASICs formés de trois sous-unités détectent les variations de l'acidité puis s'ouvrent transitoirement pour laisser entrer des ions chargés positivement dans la cellule ce qui active la signalisation neuronale. Afin de comprendre les bases moléculaires de l'activité des ASICs nous avons déterminé les sites de liaison des protons (pH-senseurs), ligands naturels des ASICs et décrit une zone importante pour l'expression fonctionnelle de ces canaux. Grâce à une validation systématique de résultats obtenus en collaboration avec l'Institut Suisse de Bioinformatique, nous avons décrit les pH-senseurs de ASICIa. Ces résultats, combinés à ceux d'autres groupes de recherche, nous ont permis de mieux comprendre comment les ASICs sont ouverts par une acidification du milieu extracellulaire. Une seconde étude souligne le rôle structural crucial d'une région conservée parmi tous les canaux ASICs : y toucher c'est diminuer l'activité de la protéine. Ce domaine permet l'harmonisation des changements dus à l'acidification du milieu extracellulaire au sein d'une même sous-unité c'est-à-dire qu'elle participe à l'induction de l'inactivation due à l'activation du canal Cette étude décrit donc quelle région de la protéine atteindre pour la bloquer efficacement en faisant une cible thérapeutique de choix.

Common Variable Immunodeficiency: molecular pathways and clinical manifestations

Common variable immunodeficiency (CVID), is a disease that is characterized by hypogammaglobulinemia as well as a defect in T, B and dendritic cells. This leads to recurrent bacterial infection mainly caused by Streptococcus pneumoniae, Klebsiella pneumoniae and Haemophilus influenzae, as well as inflammatory manifestations, i.e. granulomateous disease, gastro-intestinal disorders and chronic lung disease. Intravenous Immunoglobulin (IVIg) therapy reduces CVID susceptibility to bacterial infections to some extend. We analyzed clinical aspects of patients from our database. We recently showed that bacteria-specific CD4 T cells of CVID patients were impaired. We therefor postulated that CVID patients may harbor an acquired T-cell deficiency also called exhaustion. To test this hypothesis, we performed a comprehensive investigation of the functional profiles of bacteria-specific CD4 T cells isolated from 31 healthy individuals and 30 CVID patients. In the present study, we demonstrated that bacteria-specific but not virus-specific CD4 T cells in CVID patients harbored reduced proliferation capacity and expressed high level of PD-1. Interestingly, the blockade of PD-1/PD-1 ligands interactions restored partially bacteria but not virus-specific CD4 T-cell proliferation. Finally, we showed that 1) the level of endotoxins inversely correlates with IgG concentration, 2) IVIG treated CVID patients harbored reduced endotoxemia and 3) IgG concentration exceeding 7 mg/mL strongly reduces both the proportion of CVID patients with detectable endotoxemia and the concentration of endotoxins in plasma. Taken together our observations, suggest that primary B-cell defect(s) in CVID patients leads to recurrent bacterial infections that are associated to an acquired (secondary) impairment of CD4 T cells which may in turn exacerbate the lack of protection against extracellular bacteria.

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) plays a key role in adipocyte differentiation and insulin sensitivity. Its synthetic ligands, the thiazolidinediones (TZD), are used as insulin sensitizers in the treatment of type 2 diabetes. These compounds induce both adipocyte differentiation in cell culture models and promote weight gain in rodents and humans. Here, we report on the identification of a new synthetic PPARgamma antagonist, the phosphonophosphate SR-202, which inhibits both TZD-stimulated recruitment of the coactivator steroid receptor coactivator-1 and TZD-induced transcriptional activity of the receptor. In cell culture, SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreasing PPARgamma activity, either by treatment with SR-202 or by invalidation of one allele of the PPARgamma gene, leads to a reduction of both high fat diet-induced adipocyte hypertrophy and insulin resistance. These effects are accompanied by a smaller size of the adipocytes and a reduction of TNFalpha and leptin secretion. Treatment with SR-202 also dramatically improves insulin sensitivity in the diabetic ob/ob mice. Thus, although we cannot exclude that its actions involve additional signaling mechanisms, SR-202 represents a new selective PPARgamma antagonist that is effective both in vitro and in vivo. Because it yields both antiobesity and antidiabetic effects, SR-202 may be a lead for new compounds to be used in the treatment of obesity and type 2 diabetes.

T cell homeostasis.

The pool of mature T cells comprises a heterogeneous mixture of naive and memory CD4(+) and CD8(+) cells. These cells are long lived at a population level but differ markedly in their relative rates of turnover and survival. Here, we review how contact with exogenous stimuli, notably self MHC ligands and various gamma(c) cytokines, plays a decisive role in controlling normal T cell homeostasis.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Molecular basis of selective PPARgamma modulation for the treatment of Type 2 diabetes.

Peroxisome proliferator-activated receptors (PPARs) (alpha, beta/delta and gamma) are lipid sensors capable of adapting gene expression to integrate various lipid signals. As such, PPARs are also very important pharmaceutical targets, and specific synthetic ligands exist for the different isotypes and are either currently used or hold promises in the treatment of major metabolic disorders. In particular, compounds of the class of the thiazolinediones (TZDs) are PPARgamma agonists and potent insulin-sensitizers. The specific but still broad expression patterns of PPARgamma, as well as its implication in numerous pathways, constitutes also a disadvantage regarding drug administration, since this potentially increases the chance to generate side-effects through the activation of the receptor in tissues or cells not affected by the disease. Actually, numerous side effects associated with the administration of TZDs have been reported. Today, a new generation of PPARgamma modulators is being actively developed to activate the receptor more specifically, in a cell and time-dependent manner, in order to induce a specific subset of target genes only and modulate a restricted number of metabolic pathways. We will discuss here why and how the development of such selective PPARgamma modulators is possible, and summarize the results obtained with the published molecules.

Neofunctionalization in vertebrates: the example of retinoic acid receptors.

Understanding the role of gene duplications in establishing vertebrate innovations is one of the main challenges of Evo-Devo (evolution of development) studies. Data on evolutionary changes in gene expression (i.e., evolution of transcription factor-cis-regulatory elements relationships) tell only part of the story; protein function, best studied by biochemical and functional assays, can also change. In this study, we have investigated how gene duplication has affected both the expression and the ligand-binding specificity of retinoic acid receptors (RARs), which play a major role in chordate embryonic development. Mammals have three paralogous RAR genes--RAR alpha, beta, and gamma--which resulted from genome duplications at the origin of vertebrates. By using pharmacological ligands selective for specific paralogues, we have studied the ligand-binding capacities of RARs from diverse chordates species. We have found that RAR beta-like binding selectivity is a synapomorphy of all chordate RARs, including a reconstructed synthetic RAR representing the receptor present in the ancestor of chordates. Moreover, comparison of expression patterns of the cephalochordate amphioxus and the vertebrates suggests that, of all the RARs, RAR beta expression has remained most similar to that of the ancestral RAR. On the basis of these results together, we suggest that while RAR beta kept the ancestral RAR role, RAR alpha and RAR gamma diverged both in ligand-binding capacity and in expression patterns. We thus suggest that neofunctionalization occurred at both the expression and the functional levels to shape RAR roles during development in vertebrates.

TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95).

A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.

Cheminformatic studies of molecular properties and their influence on bioactivity in the context of drug discovery

Molecular shape has long been known to be an important property for the process of molecular recognition. Previous studies postulated the existence of a drug-like shape space that could be used to artificially bias the composition of screening libraries, with the aim to increase the chance of success in Hit Identification. In this work, it was analysed to which extend this assumption holds true. Normalized Principal Moments of Inertia Ratios (NPRs) have been used to describe the molecular shape of small molecules. It was investigated, whether active molecules of diverse targets are located in preferred subspaces of the NPR shape space. Results illustrated a significantly stronger clustering than could be expected by chance, with parts of the space unlikely to be occupied by active compounds. Furthermore, a strong enrichment of elongated, rather flat shapes could be observed, while globular compounds were highly underrepresented. This was confirmed for a wide range of small molecule datasets from different origins. Active compounds exhibited a high overlap in their shape distributions across different targets, making a purely shape­ based discrimination very difficult. An additional perspective was provided by comparing the shapes of protein binding pockets with those of their respective ligands. Although more globular than their ligands, it was observed that binding sites shapes exhibited a similarly skewed distribution in shape space: spherical shapes were highly underrepresented. This was different for unoccupied binding pockets of smaller size. These were on the contrary identified to possess a more globular shape. The relation between shape complementarity and exhibited bioactivity was analysed; a moderate correlation between bioactivity and parameters including pocket coverage, distance in shape space, and others could be identified, which reflects the importance of shape complementarity. However, this also suggests that other aspects are of relevance for molecular recognition. A subsequent analysis assessed if and how shape and volume information retrieved from pocket or respective reference ligands could be used as a pre-filter in a virtual screening approach. ln Lead Optimization compounds need to get optimized with respect to a variety of pararneters. Here, the availability of past success stories is very valuable, as they can guide medicinal chemists during their analogue synthesis plans. However, although of tremendous interest for the public domain, so far only large corporations had the ability to mine historical knowledge in their proprietary databases. With the aim to provide such information, the SwissBioisostere database was developed and released during this thesis. This database contains information on 21,293,355 performed substructural exchanges, corresponding to 5,586,462 unique replacements that have been measured in 35,039 assays against 1,948 molecular targets representing 30 target classes, and on their impact on bioactivity . A user-friendly interface was developed that provides facile access to these data and is accessible at http//www.swissbioisostere.ch. The ChEMBL database was used as primary data source of bioactivity information. Matched molecular pairs have been identified in the extracted and cleaned data. Success-based scores were developed and integrated into the database to allow re-ranking of proposed replacements by their past outcomes. It was analysed to which degree these scores correlate with chemical similarity of the underlying fragments. An unexpectedly weak relationship was detected and further investigated. Use cases of this database were envisioned, and functionalities implemented accordingly: replacement outcomes are aggregatable at the assay level, and it was shawn that an aggregation at the target or target class level could also be performed, but should be accompanied by a careful case-by-case assessment. It was furthermore observed that replacement success depends on the activity of the starting compound A within a matched molecular pair A-B. With increasing potency the probability to lose bioactivity through any substructural exchange was significantly higher than in low affine binders. A potential existence of a publication bias could be refuted. Furthermore, often performed medicinal chemistry strategies for structure-activity-relationship exploration were analysed using the acquired data. Finally, data originating from pharmaceutical companies were compared with those reported in the literature. It could be seen that industrial medicinal chemistry can access replacement information not available in the public domain. In contrast, a large amount of often-performed replacements within companies could also be identified in literature data. Preferences for particular replacements differed between these two sources. The value of combining different endpoints in an evaluation of molecular replacements was investigated. The performed studies highlighted furthermore that there seem to exist no universal substructural replacement that always retains bioactivity irrespective of the biological environment. A generalization of bioisosteric replacements seems therefore not possible. - La forme tridimensionnelle des molécules a depuis longtemps été reconnue comme une propriété importante pour le processus de reconnaissance moléculaire. Des études antérieures ont postulé que les médicaments occupent préférentiellement un sous-ensemble de l'espace des formes des molécules. Ce sous-ensemble pourrait être utilisé pour biaiser la composition de chimiothèques à cribler, dans le but d'augmenter les chances d'identifier des Hits. L'analyse et la validation de cette assertion fait l'objet de cette première partie. Les Ratios de Moments Principaux d'Inertie Normalisés (RPN) ont été utilisés pour décrire la forme tridimensionnelle de petites molécules de type médicament. Il a été étudié si les molécules actives sur des cibles différentes se co-localisaient dans des sous-espaces privilégiés de l'espace des formes. Les résultats montrent des regroupements de molécules incompatibles avec une répartition aléatoire, avec certaines parties de l'espace peu susceptibles d'être occupées par des composés actifs. Par ailleurs, un fort enrichissement en formes allongées et plutôt plates a pu être observé, tandis que les composés globulaires étaient fortement sous-représentés. Cela a été confirmé pour un large ensemble de compilations de molécules d'origines différentes. Les distributions de forme des molécules actives sur des cibles différentes se recoupent largement, rendant une discrimination fondée uniquement sur la forme très difficile. Une perspective supplémentaire a été ajoutée par la comparaison des formes des ligands avec celles de leurs sites de liaison (poches) dans leurs protéines respectives. Bien que plus globulaires que leurs ligands, il a été observé que les formes des poches présentent une distribution dans l'espace des formes avec le même type d'asymétrie que celle observée pour les ligands: les formes sphériques sont fortement sous­ représentées. Un résultat différent a été obtenu pour les poches de plus petite taille et cristallisées sans ligand: elles possédaient une forme plus globulaire. La relation entre complémentarité de forme et bioactivité a été également analysée; une corrélation modérée entre bioactivité et des paramètres tels que remplissage de poche, distance dans l'espace des formes, ainsi que d'autres, a pu être identifiée. Ceci reflète l'importance de la complémentarité des formes, mais aussi l'implication d'autres facteurs. Une analyse ultérieure a évalué si et comment la forme et le volume d'une poche ou de ses ligands de référence pouvaient être utilisés comme un pré-filtre dans une approche de criblage virtuel. Durant l'optimisation d'un Lead, de nombreux paramètres doivent être optimisés simultanément. Dans ce contexte, la disponibilité d'exemples d'optimisations réussies est précieuse, car ils peuvent orienter les chimistes médicinaux dans leurs plans de synthèse par analogie. Cependant, bien que d'un extrême intérêt pour les chercheurs dans le domaine public, seules les grandes sociétés pharmaceutiques avaient jusqu'à présent la capacité d'exploiter de telles connaissances au sein de leurs bases de données internes. Dans le but de remédier à cette limitation, la base de données SwissBioisostere a été élaborée et publiée dans le domaine public au cours de cette thèse. Cette base de données contient des informations sur 21 293 355 échanges sous-structuraux observés, correspondant à 5 586 462 remplacements uniques mesurés dans 35 039 tests contre 1948 cibles représentant 30 familles, ainsi que sur leur impact sur la bioactivité. Une interface a été développée pour permettre un accès facile à ces données, accessible à http:/ /www.swissbioisostere.ch. La base de données ChEMBL a été utilisée comme source de données de bioactivité. Une version modifiée de l'algorithme de Hussain et Rea a été implémentée pour identifier les Matched Molecular Pairs (MMP) dans les données préparées au préalable. Des scores de succès ont été développés et intégrés dans la base de données pour permettre un reclassement des remplacements proposés selon leurs résultats précédemment observés. La corrélation entre ces scores et la similarité chimique des fragments correspondants a été étudiée. Une corrélation plus faible qu'attendue a été détectée et analysée. Différents cas d'utilisation de cette base de données ont été envisagés, et les fonctionnalités correspondantes implémentées: l'agrégation des résultats de remplacement est effectuée au niveau de chaque test, et il a été montré qu'elle pourrait également être effectuée au niveau de la cible ou de la classe de cible, sous réserve d'une analyse au cas par cas. Il a en outre été constaté que le succès d'un remplacement dépend de l'activité du composé A au sein d'une paire A-B. Il a été montré que la probabilité de perdre la bioactivité à la suite d'un remplacement moléculaire quelconque est plus importante au sein des molécules les plus actives que chez les molécules de plus faible activité. L'existence potentielle d'un biais lié au processus de publication par articles a pu être réfutée. En outre, les stratégies fréquentes de chimie médicinale pour l'exploration des relations structure-activité ont été analysées à l'aide des données acquises. Enfin, les données provenant des compagnies pharmaceutiques ont été comparées à celles reportées dans la littérature. Il a pu être constaté que les chimistes médicinaux dans l'industrie peuvent accéder à des remplacements qui ne sont pas disponibles dans le domaine public. Par contre, un grand nombre de remplacements fréquemment observés dans les données de l'industrie ont également pu être identifiés dans les données de la littérature. Les préférences pour certains remplacements particuliers diffèrent entre ces deux sources. L'intérêt d'évaluer les remplacements moléculaires simultanément selon plusieurs paramètres (bioactivité et stabilité métabolique par ex.) a aussi été étudié. Les études réalisées ont souligné qu'il semble n'exister aucun remplacement sous-structural universel qui conserve toujours la bioactivité quel que soit le contexte biologique. Une généralisation des remplacements bioisostériques ne semble donc pas possible.