High expression of B-Myb (encoded by MYBL2), an oncogenic transcription factor, is associated with cell cycle deregulation and poor prognosis in several cancers, including ovarian cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adaptor for assembly of both the DREAM and MMB complexes, by a mechanism that requires the S28 phosphorylation site in LIN52. To validate our cellular findings, we determined the effect of B-Myb levels on DREAM target gene expression in HGSOC tissue samples and corresponding patient outcomes. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification and establish the rationale for targeting B-Myb to restore cell cycle control.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is a genetic disorder that results from triplication of human chromosome 21 (Hsa21) and occurs in around 1 in 1000 live births. All individuals with DS present with skeletal abnormalities typified by craniofacial features, short stature and low bone mineral density (BMD). Differences between males and females with DS suggest a sexual dimorphism in how trisomy affects skeletal deficits associated with trisomy 21 (Ts21). Previous investigations of skeletal abnormalities in DS have varied methodology, sample sizes and ages making the underlying causes of deficits uncertain. Mouse models of DS were used to characterize skeletal abnormalities, but the genetic and developmental origin remain unidentified. Over-expression Dyrk1a, found on Hsa21 and mouse chromosome 16 (Mmu16) has been linked to cognitive deficits and skeletal deficiencies. Dp1Tyb mice contain three copies of all of the genes on Mmu16 that are homologous to Hsa21, males and females are fertile, and therefore are an excellent model to test the hypothesis that gene dosage influences the sexual dimorphism of bone abnormalities in DS. Dp1Tyb at 6 weeks 16 weeks showed distinctive abnormalities in BMD, trabecular architecture, and reduced bone strength over time that occur generally through an interaction between sex and genotype. Increased gene dosage and sexual dimorphism in Dp1Tyb mice revealed distinct phenotypes in bone formation and resorption. To assess how Dyrk1a influences the activity and function of osteoblasts Ts65Dn female trisomic mice, female mice with a floxed Dyrk1a gene (Ts65Dn, Dyrk1afl/+) were be bred to Osx1-GFP::Cre+ mice to generate Ts65Dn animals with a reduced copy of Dyrk1a in mature osteoblast cells. Female Ts65Dn,Dyrk1a+/+/+ and Ts65Dn,Dyrk1a+/+/-displayed significant defects in both trabecular architecture and cortical geometry. Ultimate force was reduced in trisomic animals, suggesting whole bone and tissue level properties are not adversely affected by trisomy. Reduction of Dyrk1a functional copy number in female mice did not improve skeletal deficits in an otherwise trisomic animal. Dyrk1a may not alter osteoblast cellular activity in an autonomous manner in trisomic female mice. These data establish sex, gene dosage, skeletal site and age as important factors in skeletal development of the skeleton in DS mice, potentially paving the way for identification of the causal dosage-sensitive genes in both male and female animals.

Au cours de cette thèse, deux sujets distincts ont été étudiés : d’une part la synthèse d’analogues de la coelentérazine, substrat de différentes luciférases, pour une application en imagerie in vivo, et d’autre part la synthèse d’analogues du (-)-EGCG, inhibiteur de la kinase Dyrk1a, impliquée notamment dans les troubles cognitifs rencontrés dans la trisomie 21.La problématique du premier sujet consistait à obtenir des analogues de la coelentérazine conservant leur activité sur deux luciférases, la luciférase Renilla et l’aequorine, tout en induisant un déplacement vers le rouge de la bioluminescence produite par ces enzymes. L’aequorine, sensible au calcium, représentait la cible biologique principale du projet.Sept analogues dont six originaux ont été obtenues par des méthodes de synthèse classiques, et leurs activités ont pu être testées sur les deux luciférases choisies, avec des résultats probants : malgré des émissions de lumière moins intenses que celles obtenues avec la coelentérazine native, plusieurs molécules ont entrainé un déplacement vers le rouge de la longueur d’onde d’émission de lumière par bioluminescence allant jusqu’à 27 nm pour l’aequorine et plus de 120 nm pour la luciférase Renilla.Le second sujet, de chimie médicinale classique, a principalement consisté à la synthèse d’analogues du gallate d’épigallocatéchine (EGCG) au squelette simplifié, et au sein desquels le cycle pyranique caractéristique des catéchines a été remplacé par un carbocycle. Plusieurs molécules ont pu être synthétisées, dont deux présentant le motif hexaphénol. Leur activité inhibitrice de Dyrk1a a pu être testée in vitro et l’une d’entre elle s’est déjà révélée plus active que l’EGCG. / During this thesis, two distinct projects were studied: on the one hand, the synthesis of coelenterazine analogs, substrate of several luciferases, in the purpose of using them for in vivo imaging, and on the other, the synthesis of (-)-EGCG analogs, inhibitor of the Dyrk1a kinase, which interests us for the role it plays in the mental retardation existing in the Down Syndrome disease.The problematic of the first project consisted in obtaining coelenterazine analogs that would not only maintain their activity on two luciferases, the Renilla luciferase and aequorine, but they should also induce a red-shift of the bioluminescence produced by these enzymes. Because of its sensitivity to calcium, aequorine was the main biologic target of this project.Seven analogs, of which six had an original structure, were synthesized through usual synthetic methodologies and their activities on both aequorine and Renilla luciferase were tested in vitro, with interesting results: even if the intensities of light emission were weaker than those obtained with native coelenterazine, several molecules produced a red-shift of the emission wavelength of bioluminescence, up to 27nm for aequorine and more than 120nm for the Renilla luciferase. The second project, of classical medicinal chemistry, mainly consisted in the synthesis of epigallocatechin gallate analogs (EGCG) with a simplified backbone and in which the pyranic ring typical of catechins was replaced by a carbocycle. Several molecules were synthesized, two of them possessing the hexaphenol motif. Their inhibiting activity of Dyrk1a was tested in vitro and one already showed a better activity than natural EGCG.

DYRK1A es uno de los principales genes candidatos que podrían explicar algunos de los defectos neurológicos asociados al fenotipo Síndrome de Down (SD); desde el retraso mental, rasgo común a todos los individuos con SD hasta los déficits motores, también muy frecuentes entre la población con SD. Con el fin de validar la implicación de DYRK1A en el fenotipo SD se ha desarrollado una estrategia de terapia génica basada en la reducción de la expresión del gen mediante interferencia del RNA, en el modelo transgénico TgDyrk1A, y se han evaluado los efectos en el fenotipo motor de estos animales. Además se ha estudiado la implicación de los receptores glutamatérgicos de tipo NMDA en las alteraciones motoras descritas en el modelo. Los resultados obtenidos en este trabajo ponen de manifiesto la validez de la estrategia desarrollada y apuntan a una desregulación de los receptores de NMDA como uno de los mecanismos moleculares subyacentes de las disfunción motora presente en el modelo TgDyrk1A. / The are growing evidences to consider DYRK1A as a candidate gene for some of the neurological alterations present in DS phenotype such as mental retardation which is a common feature in the syndrome, or motor deficits which show a high prevalence among DS individuals. With the aim to validate the contribution of Dyrk1A to DS phenothype, we have developped a gene therapy strategy based on RNA interference to reduce gene expression in the transgenic model TgDyrk1A, and we have evaluated the effects in the motor phenotype of these animals. Moreover, we have studied the implication of the NMDA glutamate receptor in the motor alterations present in the model. The results obtained validate the strategy developped and suggest the deregulation of the NMDA receptor as one of the main causes underlying motor dysfunction in TgDyrk1A mice.

Les effets du dérèglement de l’expression des protéines kinases DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) et plus particulièrement de DYRK1A sont étudiées dans le cas des maladies neurodégénératives (maladie d’Alzheimer, syndrome de Down) et celui de certains cancers. Ces travaux de thèse s’inscrivent dans la continuité des acquis du laboratoire et des résultats des évaluations de l’activité inhibitrice obtenus sur les thiazolo[5,4-f]quinazolines et les thiazolo[5,4-f]quinazolin-9(8H)-ones. L’objectif principal est la modulation du groupement en position C2 des thiazolo[5,4-f]quinazolin-9(8H)-ones en faisant varier la taille et la nature des substituants afin de former des inhibiteurs potentiels sélectifs de DYRK1A. Le premier chapitre de ce manuscrit concerne l’addition de nucléophiles originaux tels que des acides aminés sur la fonction nitrile en position C2 de la 8-benzylthiazolo[5,4-f]quinazolin-9(8H)-one. Le second chapitre décrit l’optimisation de la synthèse des thiazolo[5,4-f]quinazolin-9(8H)-ones ainsi que l’arylation directe ces motifs. La réaction de débenzylation des dérivés C2-arylés et la fonctionnalisation par C–H alcénylation de la 8-benzylthiazolo[5,4-f]quinazolin-9(8H)-one sont aussi décrites dans cette partie. Le troisième chapitre concerne l’évaluation de l’activité biologique de la plupart des composés synthétisés au cours de ces travaux, révélant le FC162 en tant qu’inhibiteur de DYRK1A. Ces résultats ont conduit à la modulation en position C2 de motifs plus simples : les benzo[d]thiazoles, permettant d’étudier la régiosélectivité du 6-aminobenzo[d]thiazole-2,7-dicarbonitrile et l’arylation directe de ces dérivés bicycliques. / The effects of expression modulation of protein kinases DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) and especially of DYRK1A are characterized in a variety of diseases including neurodegenerative diseases (Alzheimer’s disease or Down syndrome) and cancers. This thesis deals with our laboratory knowledge and previous results obtained with thiazolo[5,4-f]quinazolines and thiazolo[5,4-f]quinazolin-9(8H)-ones and their activity. The main part of this thesis is focused on the C2-modulation of thiazolo[5,4-f]quinazolin-9(8H)-ones to synthesize potent DYRK1A inhibitors. The first chapter of this manuscript describes the addition of nucleophiles such as amino acids at the C2-function of 8-benzylthiazolo[5,4-f]quinazolin-9(8H)-ones. The second chapter is focused on the optimization of the thiazolo[5,4-f]quinazolin-9(8H)-ones synthesis and their C–H arylation. Deprotection of C2-arylated products and C–H alkenylation approach of 8-benzylthiazolo[5,4-f]quinazolin-9(8H)-one are also described. The third chapter deals with the inhibitory activity evaluation of most of the compounds prepared along this work shown FC162 as DYRK1A inhibitor. These results led to C2 modulation of simpler structures such as benzo[d]thiazoles by studying the C–H arylation of these bicyclic derivatives.

<p>Down syndrome (DS) is a genetic disorder that results from triplication of human chromosome 21 (Hsa21) and occurs in around 1 in 1000 live births. All individuals with DS present with skeletal abnormalities typified by craniofacial features, short stature and low bone mineral density (BMD). Differences between males and females with DS suggest a sexual dimorphism in how trisomy affects skeletal deficits associated with trisomy 21 (Ts21). Previous investigations of skeletal abnormalities in DS have varied methodology, sample sizes and ages making the underlying causes of deficits uncertain. Mouse models of DS were used to characterize skeletal abnormalities, but the genetic and developmental origin remain unidentified. Over-expression <i>Dyrk1a</i>, found on Hsa21 and mouse chromosome 16 (Mmu16) has been linked to cognitive deficits and skeletal deficiencies. Dp1Tyb mice contain three copies of all of the genes on Mmu16 that are homologous to Hsa21, males and females are fertile, and therefore are an excellent model to test the hypothesis that gene dosage influences the sexual dimorphism of bone abnormalities in DS. Dp1Tyb at 6 weeks 16 weeks showed distinctive abnormalities in BMD, trabecular architecture, and reduced bone strength over time that occur generally through an interaction between sex and genotype. Increased gene dosage and sexual dimorphism in Dp1Tyb mice revealed distinct phenotypes in bone formation and resorption. To assess how <i>Dyrk1a</i> influences the activity and function of osteoblasts Ts65Dn female trisomic mice, female mice with a floxed <i>Dyrk1a</i> gene (Ts65Dn, <i>Dyrk1a</i>^fl/+) were be bred to <i>Osx1</i>-GFP::Cre+ mice to generate Ts65Dn animals with a reduced copy of <i>Dyrk1a </i>in mature osteoblast cells. Female Ts65Dn,<i>Dyrk1a^+/+/+</i>and Ts65Dn,<i>Dyrk1a^+/+/-</i>displayed significant defects in both trabecular architecture and cortical geometry. Ultimate force was reduced in trisomic animals, suggesting whole bone and tissue level properties are not adversely affected by trisomy. Reduction of <i>Dyrk1a</i> functional copy number in female mice did not improve skeletal deficits in an otherwise trisomic animal. <i>Dyrk1a </i>may not alter osteoblast cellular activity in an autonomous manner in trisomic female mice. These data establish sex, gene dosage, skeletal site and age as important factors in skeletal development of the skeleton in DS mice, potentially paving the way for identification of the causal dosage-sensitive genes in both male and female animals. </p>

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the result of trisomy of human chromosome 21 (Hsa 21) and occurs in approximately 1/700 live births. Mouse models of DS have been crucial in understanding the gene-phenotype relationships that underlie many DS anomalies. The Ts65Dn mouse model, trisomic for half of the Hsa 21 orthologs replicates many DS phenotypes including craniofacial alterations such as a small, dysmorphic mandible, midface, and maxilla. Other mouse models, such as the Ts1Rhr which contains a triplication of 33 Hsa 21 orthologs, have been used to better understand the genes responsible for craniofacial alterations. Our laboratory has demonstrated that the postnatal mandibular phenotype found in Ts65Dn mice can be traced back to an original neural crest cell (NCc) deficit in the developing first pharyngeal arch (PA1) at embryonic day 9.5 (E9.5). Furthermore, evidence suggested that both a proliferation deficit in the PA1 and a migration deficit in the NCC from the neural tube (NT) could be the mechanism behind this deficit. However, the molecular mechanisms behind these deficits remain to be elucidated. Due to the involvement of the Hsa 21 genes DYRK1A and RCAN1 in regulation of signaling pathways including NFATc (NFAT2), a transcription factor known to influence cellular proliferation and, later, bone development, we hypothesized that dysregulation of these genes could underlie the cellular deficit in the PA1. Furthermore, we hypothesized that targeting Dyrk1a by decreasing activity or available protein could ameliorate the established deficits. Through the use of RNA isolation techniques and cell culture systems of cell from the PA1 and NT of E9.5 Ts65Dn, Ts1Rhr, and control embryos, we established that trisomic genes Dyrk1a and Rcan1 ara dysregulated in both structures and that these two genes may interact. Furthermore, we established that a proliferation deficit in the Ts65Dn PA1 and a migration deficit in the Ts65Dn PA1 and NT exists at E9.5 and can be rescued to euploid levels in vitro with the addition of the Dyrk1a inhibitor, EGCG, a green tea polyphenol. We also confirmed that harmine, a more highly studied and specific Dyrk1a inhibitor, is capable of similar effects on proliferation of PA1 cell from E9.5 Ts65Dn embryos. Furthermore, when Ts65Dn pregnant mothers were treated with EGCG in vivo, the cellular deficit found in the developing E9.5 embryonic PA1 was rescued to near euploid volume and NCC number. Treatment with EGCG did not adversely impact litter size or embryonic development. Interestingly, euploid embryonic volume increased with EGCG treatment. Expression analysis of the E9.5 PA1 of EGCG treated Ts65Dn and control embryos revealed dysregulation of several genes involved in craniofacial and developmental pathways including Dyrk1a, Rcan1, Ets2 and members of the sonic hedgehog pathways. Our novel results provide a foundation for better understanding the molecular mechanisms of craniofacial development and may provide evidence-based therapeutic options to improve the quality of life for individuals with DS.

Depuis la mise en évidence de l’existence des protéines kinases vers la fin des années 1950 cette famille d’enzymes s’est vu attribuer d’importants rôles dans divers mécanismes pathologiques notamment dans des processus de cancérisations. Plus récemment ces enzymes ont été identifiées comme potentiellement impliquées dans d’autres types de maladies telles que les maladies neurodégénératives.Deux projets de recherche seront présentés. Le premier projet expose la conception et la synthèse de nouveaux composés tricycliques de la famille des pyrido[3,4-g]quinazolines. Les propriétés inhibitrices de kinases des premiers dérivés ont été évaluées sur un panel de cinq kinases (CDK5, CK1, GSK3, CLK1 and DYRK1A) connues pour leurs implications dans la maladie d’Alzheimer. L’intérêt de ces nouveaux squelettes tricycliques comme inhibiteurs de kinases a été validé par des activités inhibitrices nanomolaire à l’encontre des kinases DYRK1A et CLK1. D’autre part l’obtention de structures co-crystallographiques d’interaction de deux dérivés avec le site ATP de la kinase CLK1 a permis de rationnaliser la substitution du motif pyrido[3,4-g]quinazoline. Le second projet présente le développement d’un nouveau dérivé de la staurosporine aglycone (K252c) dans lequel la partie lactame a été remplacée par un noyau pyrazole. Une étude préliminaire des propriétés biologiques de l’indolopyrazolocarbazole obtenu met en avant une cytotoxicité, du même ordre de grandeur que K252c, contre les lignées cellulaires K562 (leucémie humaine) et HCT116 (carcinome du colon). En revanche, le composé chef de file s’est révélé être un faible inhibiteur de cibles connues de K252c, les isoformes α and γ de la protéine kinase C et présente un bon potentiel inhibiteur des kinases Pim 1-3. Ce nouveau chemotype pourrait être un inhibiteur de kinases prometteur. / In 1950’s protein kinases were found to play a critical role in cell signaling, rising strong research potential for this enzyme family. Initially investigated for their implications in cancerogenesis they were more recently found to be involved in a wide variety of diseases including neurodegenerative pathologies. Herein will be presented two research projects that offer bright new perspectives for the inhibition of kinases involved whether in neurodegenerative diseases or cancers.First, the design and synthesis of new pyrido[3,4-g]quinazoline derivatives will be described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A) that are known to play a potential role in Alzheimer’s disease. The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/ DYRK1A activity was validated by nanomolar potencies. CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket and led to the synthesis of new diversely substituted pyrido[3,4-g]quinazolines.Then the synthesis of a new derivative of the staurosporine aglycon (K252c), in which the lactam ring was replaced by a pyrazole moiety, will be depicted. The resulting indolopyrazolocarbazole inhibited Pim isoforms 1–3 whereas it did not impair the activity of two known targets of K252c, protein kinase C isoforms α and γ . The lead compound exhibited same cytotoxic activity as K252c toward both human leukemia and colon carcinoma cell lines (K562 and HCT116), strongly suggesting that this new scaffold deserves further investigations for treatment of malignancies associated with kinases activities.

PolyHistidine repeats and DYRK1A: from the localization to the functionEl principal objectiu d'aquesta tesi ha estat el d'esbrinar noves funcions de la proteína quinasa DYRK1A en el nucli cel.lular. Donat que el domini de repetició d'histidines de DYRK1A dirigeix la proteína al compartiment d'speckles nuclears, aquesta propietat ha estat utilitzada per adreçar aquesta pregunta. Els resultats obtinguts en aquesta tesi han permès proposar els homopolímers d'histidina com una nova i general senyal de localització a speckles nuclears. Proteïnes amb segments de polihistidines, la majoria d'elles factors de transcripció, mostren un comportament intranuclear dinàmic, compatible amb un model en el quèl diferents dominis d'interacció competeixen entre ells pel reclutament de la proteína a diferents subcompartiments nuclears. El mecanisme molecular que media l'acumulació a speckles de les proteïnes amb polihistines s'ha estudiat utilitzant DYRK1A com a model. Els resultats obtinguts exclouen la unió a l'RNA com a mecanisme de reclutament i concloure que, aquest, ocorre mitjançant la interacció amb proteïnes residents. S'han identificat dues noves proteïnes interactores per a DYRK1A, l'RNA polimerasa II i el factor de transcripció Brn-3b. La fosforilació de DYRK1A sobre el domini C-terminal o CTD de l'RNA polimerasa II suggereix una funció directa de la quinasa en el procés de transcripció o del seu acoblament al processament d'RNAs missatgers. La fosforilació de DYRK1A sobre el domini d'activació de Brn-3b sembla regular positivament l'activitat transcripcional d'aquest factor. Aquests resultats indiquen una funció activa de DYRK1A en la regulació de la transcripció gènica, tant directament sobre la maquinària transcripcional com indirectament, modulant l'activitat de factors de transcripció. PolyHistidine repeats and DYRK1A: from the localization to the functionThe main objective of this thesis work has been to identify new roles for the protein kinase DYRK1A in the cell nucleus. Given that a histidine repeat in DYRK1A targets the protein to the nuclear speckle compartment, this property has been used as a tool to approach the question. The results obtained in this thesis work have allowed proposing homopolymeric histidine runs as a novel and general nuclear speckle-directing signal. Proteins with polyHistidine segments, mostly transcription factors, present a dynamic intranuclear behaviour compatible with a model in which distinct interacting domains compete for recruiting elements within the nucleus. The molecular mechanisms that mediate speckle accumulation have been studied in DYRK1A as a model system. The results allow excluding RNA binding as the recruiting mechanism and concluding that targeting is mediated by interaction with speckle-resident proteins. Two novel DYRK1A interactors have been identified during the study, the RNA polymerase II and the transcription factor Brn-3b. DYRK1A phosphorylation of the C-terminal domain or CTD of the RNA polymerase II suggests a direct role of DYRK1A on transcription or coupling of transcription with RNA processing. DYRK1A phosphorylation of Brn-3b within its activation domain seems to positively regulate Brn-3b transcriptional activity. These results confirm an active role for DYRK1A in gene transcription regulation both direct on the transcriptional machinery and indirect by modulating the activity of transcription factors.

La protéine kinase DYRK1A fait partie du groupe des CMGC kinases et est impliquée dans divers processus neurodégénératifs tels que la maladie d’Alzheimer.Dans ce cadre, une étude topologique a été menée autour d’un hit imidazo[1,2-B]pyridazine identifié par un criblage biologique. Ce composé a servi à concevoir des inhibiteurs ATP-Compétitifs de DYRK1A par l’utilisation de méthodes métallo-Catalysées (Pd, Cu) pour introduire divers fragments fonctionnalisés.Sur les soixante dérivés imidazo[1,2-X]azine synthétisés, sept composés ont montré une affinité nanomolaire pour DYRK1A (IC50 = 41-130 nM). En parallèle de ce travail de pharmacochimie, le développement de nouvelles méthodologies de synthèse a visé à la polysubstitution régiosélective du cycle pyridazine.En dernier lieu, nous avons donné les éléments et concepts permettant la construction de chimiothèques virtuelles dérivées de pyridazines et destinées à être criblées in silico. / DYRK1A protein kinase belongs to the CMGC group and is involved in neurodegenerative disorders such as Alzheimer’s disease.In this context we examined an imidazo[1,2-B]pyridazine hit identified by biological screening, through detailed structure-Activity relationship studies. This compound was used to synthesize DYRK1A ATP-Competitive inhibitors by using metallo-Catalyzed methodologies (Pd, Cu) in order to introduce various functionalized moieties.Out of the 60 derivatives synthesized, 7 compounds showed nanomolar activities (IC50 = 41-130 nM).Beside this work of medicinal chemistry, new synthetic methodologies has been developed to regioselectively access polysubstituted pyridazine derivatives. Finally, we developed data and concepts to establish virtual pyridazine libraries for in silico screening.