京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20243号 / 医博第4202号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 原田 浩, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le sujet de cette thèse porte sur la synthèse d'inhibiteurs spécifique de la Sérine-Thréonine phosphatase I (PP1). Un criblage de la chimiothèque de l'institut Curie, réalisé par l'équipe du Dr. Popov a permis d'identifier une 3-aryl-1(2H)isoquinolone, qui perturbe la dynamique des microtubules et qui s’est ensuite avéré être un inhibiteur sélectif de PP1. Dans une première partie, nous avons mis au point une nouvelle méthodologie de synthèse de ces composés hétérocycliques par une réaction tandem d’aminocarbonylation-cyclisation pallado catalysée. L’étude d’une seconde voie de synthèse de ces composés a été étudié par réaction d'arylation direct d'une 1(2H)isoquinolone. Dans le but de trouver d’autres hit, ligand de cette phosphatase, nous avons tenté de développer un test de triple hybride chimique, en collaboration avec la société Hybrigenics. Ce test est basé sur l’interaction de notre inhibiteur hit avec la phosphatase PP1. Pour cela, nous avons synthétisé une sonde à partir de la molécule hit initiale. La deuxième partie a trait à un développement de chimie médicinal pour optimiser le hit initial. Des dérivés de très bonne sélectivité pour l’enzyme cible ont été préparés. / This PhD thesis deals with the synthesis of serine threonine phosphatase I (PPI) inhibitors. This project started with the screening of the Institut Curie’s Library carried out by Dr. Popov team. They identified a 3-aryl-1(2H)isoquinolone (hit molecule) which strongly disturbs the microtubules dynamics. In the first part, we designed an original methodology to prepare those heterocycles, though a tandem palladium catalyzed aminocarbonylation/cyclization reaction. Then, we studied the direct arylation reaction to obtain the desired scaffold. In collaboration with Hybrigenics, we synthesize a probe for a triple hybrid system, based on the specific interaction of the hit molecule with its target PPI. Thanks to this system, one could identify new inhibitors of the targeted phosphatase protein. Eventually, a library of isoquinolones derivatives was synthesized. During the invitro tests, some of those molecules proved to be very specific for the serine threonine phosphatase I.

Protein phosphorylation constitutes an important mechanism for cellular regulation in both Eucarya and Bacteria. All living organisms evolved from a common progenitor; this implies that protein phosphorylation as a means of regulation also exists in Archaea. Previously, in the sulfur-dependent archaeon Sulfolobus solfataricus a gene was cloned encoding a protein-serine/threonine phosphatase that was similar to eucaryal protein-serine/threonine phosphatases type 1, 2A, and 2B. To identify protein phosphatases in other archaeons, oligonucleotides encoding conserved regions of eucaryal protein-serine/threonine phosphatases were used in the polymerase chain reaction to amplify genomic DNA from the methanogenic archaeon Methanosarcina thermophila. From the PCR reaction a fragment of DNA was isolated that encoded a portion of a protein phosphatase. Using this DNA fragment as a probe, the entire phosphatase gene was isolated. The amino acid sequence of the phosphatase encoded by this gene displayed greater than 30% identity with eucaryal protein-serine/threonine phosphatase type 1. The gene encoding the Methanosarcina phosphatase was expressed in Escherichia coli. The expressed protein exhibited protein serine phosphatase activity that was sensitive to inhibitors of eucaryal phosphatases such as okadaic acid, microcystin, calyculin, and tautomycin. In order to identify potential endogenous substrates of archaeal protein-serine/threonine phosphatases and kinases, a study was initiated to characterize the most prominent phosphoproteins in S. solfataricus. Cell extracts were incubated with [γ-³²P] ATP, MgCl₂, and MnCl₂, and the proteins in the extracts were separated by SDS-PAGE. Autoradiography of the gels revealed four prominent phosphoproteins with apparent molecular masses of 35, 46, and 50 kDa. N-terminal sequence analysis and enzymatic assays of the 35 kDa phosphoprotein identified this phosphoprotein as the a-subunit of succinyl-CoA synthetase. N-terminal sequence analysis and enzymatic assays revealed that the 50 kDa phosphoprotein was a hexosephosphate mutase. Neither the 50 kDa nor the 35 kDa phosphoprotein appeared to be the target of protein kinases or phosphatases. Therefore, while protein-serine phosphatases exist in Archaea, the targets of these phosphatases have yet to be determined. / Ph. D.

A proteína serina/treonina fosfatase do tipo 1 (PP1) é a principal serina/treonina fosfatase envolvida na regulação de diversos processos tais como metabolismo, crescimento e divisão celular, síntese protéica e processamento de RNA. A holoenzima PP1 é constituída de uma subunidade catalítica conservada (PP1c) e subunidades reguladoras variáveis. Em mamíferos já foram identificados dezenas de polipeptídeos que associam-se direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre as proteínas que se associam a PP1c, muitas têm função inibitória como o inibidor-1 (I-1) e o inibidor-2 (I-2). A partir de extratos de micélios de Neurospora crassa foi purificada uma proteína, denominada INc-1, que atua in vitro como inibidor da atividade de fosforilase fosfatase de PP1c e constitui-se no primeiro exemplo de subunidade reguladora da PP1 descrito em fungos filamentosos. INc-1 apresenta diversas características bioquímicas comuns ao I-2 de mamíferos. Seqüências parciais de aminoácidos de três fragmentos proteolíticos obtidos de INc-1 permitiram a identificação de uma ORF (fase aberta de leitura) no genoma de N. crassa que provavelmente codifica INc-1. A análise dessa ORF mostrou que a sequência de aminoácidos do INc-1 é similar a do I-2, especialmente em regiões supostamente envolvidas em sua interação com a PP1c. Neste trabalho descrevemos a clonagem e a expressão em bactérias da sequência codificadora de INc-1. A atividade inibidora de PP1c de duas isoformas recombinantes purificadas, INc-1L e INc-1, foram avaliadas e comparadas. A forma denominada INc-1L apresenta em sua região aminoterminal um segmento de 38 aminoácidos derivado da retenção de um íntron, sem alterar a fase de leitura. Ambas proteínas recombinantes exibiram efeito inibidor sobre a atividade de fosforilase fosfatase de PP1c recombinante, sendo que a IC50 determinada para INc-1L foi de ~50nM e para INc-1 foi de ~11nM, sugerindo que a retenção do segmento de aminoácidos codificado pelo íntron na isoforma INc-1L diminui seu potencial inibitório. Verificamos também que o mRNA de INc-1 é expresso durante o crescimento vegetativo de N.crassa, apresentando níveis máximos na fase exponencial. / Type 1 protein serine/threonine phosphatases (PP1) play important roles in the regulation of many cellular functions including metabolism, cell growth and division, protein synthesis and pre-mRNA splicing. PP1 holoenzyme consists of one highly conserved catalytic subunit (PP1c) and variable regulatory subunits. A number of proteins that interact with PP1c have been described in mammals and the respective holoenzymes present distinct substrate specificity and/or different subcelular localization. Among the proteins that interact with PP1c, there are many with inhibitory effect such as inhibitor-1 (I-1) and inhibitor-2 (1-2). It has been demonstrated that a protein denominated INc-1, purified from Neurospora crassa extracts, specifically inhibits PP1c and has biochemical properties that resemble those of mammalian I-2. INc-1 is the first example of a PP1c regulatory subunit in filamentous fungi. Partial amino acid sequences of INc-1 led to the identification of an ORF (open reading frame) in Neurospora crassa genome which appears to encode INc-1. This ORF shows similarity with mammalian I-2 mainly in regions mapped as sites for interaction with PP1c. In this work we report the cloning and bacterial expression of the coding sequence for INc-1. The PP1c inhibitory activities of two recombinant isoforms, named INc-1L and INc-1, were compared. INc-1L aminoacid sequence presents an in frame segment of 38 residues encoded by an non-processed intron. 80th recombinant proteins showed inhibitory effect against phosphorylase phosphatase activity of recombinant PP1c, with IC50 of ~50nM for INc-1L and ~11nM for INc-1, suggesting that retention of the 38 residue segment decrease the inhibitory potential of INc-1L. We have also verified that INc-1 mRNA is expressed during N.crassa vegetative growth with maximum level at the exponential phase.

A proteína serina/treonina fosfatase do tipo 1 (PP1) é a principal serina/treonina fosfatase envolvida na regulação de diversos processos tais como metabolismo, crescimento e divisão celular, síntese protéica e processamento de RNA. A holoenzima PP1 é constituída de uma subunidade catalítica conservada (PP1c) e subunidades reguladoras variáveis. Em mamíferos já foram identificados dezenas de polipeptídeos que associam-se direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre as proteínas que se associam a PP1c, muitas têm função inibitória como o inibidor-1 (I-1) e o inibidor-2 (I-2). A partir de extratos de micélios de Neurospora crassa foi purificada uma proteína, denominada INc-1, que atua in vitro como inibidor da atividade de fosforilase fosfatase de PP1c e constitui-se no primeiro exemplo de subunidade reguladora da PP1 descrito em fungos filamentosos. INc-1 apresenta diversas características bioquímicas comuns ao I-2 de mamíferos. Seqüências parciais de aminoácidos de três fragmentos proteolíticos obtidos de INc-1 permitiram a identificação de uma ORF (fase aberta de leitura) no genoma de N. crassa que provavelmente codifica INc-1. A análise dessa ORF mostrou que a sequência de aminoácidos do INc-1 é similar a do I-2, especialmente em regiões supostamente envolvidas em sua interação com a PP1c. Neste trabalho descrevemos a clonagem e a expressão em bactérias da sequência codificadora de INc-1. A atividade inibidora de PP1c de duas isoformas recombinantes purificadas, INc-1L e INc-1, foram avaliadas e comparadas. A forma denominada INc-1L apresenta em sua região aminoterminal um segmento de 38 aminoácidos derivado da retenção de um íntron, sem alterar a fase de leitura. Ambas proteínas recombinantes exibiram efeito inibidor sobre a atividade de fosforilase fosfatase de PP1c recombinante, sendo que a IC50 determinada para INc-1L foi de ~50nM e para INc-1 foi de ~11nM, sugerindo que a retenção do segmento de aminoácidos codificado pelo íntron na isoforma INc-1L diminui seu potencial inibitório. Verificamos também que o mRNA de INc-1 é expresso durante o crescimento vegetativo de N.crassa, apresentando níveis máximos na fase exponencial. / Type 1 protein serine/threonine phosphatases (PP1) play important roles in the regulation of many cellular functions including metabolism, cell growth and division, protein synthesis and pre-mRNA splicing. PP1 holoenzyme consists of one highly conserved catalytic subunit (PP1c) and variable regulatory subunits. A number of proteins that interact with PP1c have been described in mammals and the respective holoenzymes present distinct substrate specificity and/or different subcelular localization. Among the proteins that interact with PP1c, there are many with inhibitory effect such as inhibitor-1 (I-1) and inhibitor-2 (1-2). It has been demonstrated that a protein denominated INc-1, purified from Neurospora crassa extracts, specifically inhibits PP1c and has biochemical properties that resemble those of mammalian I-2. INc-1 is the first example of a PP1c regulatory subunit in filamentous fungi. Partial amino acid sequences of INc-1 led to the identification of an ORF (open reading frame) in Neurospora crassa genome which appears to encode INc-1. This ORF shows similarity with mammalian I-2 mainly in regions mapped as sites for interaction with PP1c. In this work we report the cloning and bacterial expression of the coding sequence for INc-1. The PP1c inhibitory activities of two recombinant isoforms, named INc-1L and INc-1, were compared. INc-1L aminoacid sequence presents an in frame segment of 38 residues encoded by an non-processed intron. 80th recombinant proteins showed inhibitory effect against phosphorylase phosphatase activity of recombinant PP1c, with IC50 of ~50nM for INc-1L and ~11nM for INc-1, suggesting that retention of the 38 residue segment decrease the inhibitory potential of INc-1L. We have also verified that INc-1 mRNA is expressed during N.crassa vegetative growth with maximum level at the exponential phase.

La cohésion des chromatides sœurs est assurée par un complexe protéique en forme d’anneau assurant leur capture topologique. Ce complexe est constitué par des protéines conservées de la levure à l’Homme regroupées sous le terme « cohésine » : Smc1, Smc3 et la phosphoprotéine Scc1 fermant l’anneau (respectivement Psm1, Psm3 et Rad21 chez Schizosaccharomyces pombe). Les protéines régulatrices Rad61-Wapl, Pds5 et Scc3 (Wpl1,Pds5 et Psc3 respectivement chez S. pombe) interagissent avec l’anneau via Scc1. Il a été proposé que la capture de l’ADN par les cohésines nécessite l’ouverture transitoire de l’interface Smc1/Smc3. La réaction de dissociation fait quant à elle intervenir le sous-complexe Wapl/Pds5/Scc3 entraînant vraisemblablement l’ouverture de l’interface Scc1/Smc3. Le mécanisme par lequel la cohésion est créée et celui par lequel Wapl promeut la dissociation des cohésines des chromosomes, sont encore inconnus. Parmi les mutants de cohésion chez Saccharomyces cerevisiae, la mutation thermosensible eco1-1 affecte le gène ECO1 codant une acétyl-transférase, essentielle à la viabilité cellulaire, conservée de la levure à l’Homme (Eco1 « Establishment of Cohesion » chez S. cerevisiae, Eso1 chez S.pombe, ESCO1-2 chez l’Homme) et ayant Smc3 pour substrat. Il a été montré que l’acétyl-transférase s’oppose à l’action de dissociation de Wapl. C’est un crible génétique réalisé par plusieurs équipes, visant à trouver des mutants suppresseurs d’eco1-1, qui a permis d’identifier les gènes codant les protéines Wapl, Pds5, Scc3 et Smc3 comme composants du mécanisme d’ouverture de l’anneau de cohésine. Un crible similaire a été réalisé chez S.pombe dans notre laboratoire, dans le but de trouver des suppresseurs de la mutation thermosensible eso1-H17. Ce crible a identifié les gènes orthologues à ceux trouvés chez la levure : wpl1, pds5, psc3 et psm3 mais aussi le gène codant la sous-unité catalytique du complexe sérine/thréonine phosphatase de type IV (PP4), noté pp4c. Nous avons alors mis en œuvre des expériences pour caractériser PP4c ainsi que sa sous-unité régulatrice Psy2 qui s’est révélée être également impliquée dans la cohésion des chromatides soeurs. Nous avons également identifié la protéine Rad21 comme substrat du complexe PP4, puis identifié les phosphosites potentiellement cibles de PP4, pour ensuite cribler et analyser des phosphomutants de Rad21 récapitulant l’effet suppresseur de la délétion de PP4. / Sister-chromatid cohesion is ensured by a ring shape protein complex which is in charge of their topological embrace. This complex consists of proteins which are conserved from yeast to human and grouped under the term “cohesin”: Smc1, Smc3 and the phosphoprotein Scc1 which closes the ring (respectively Psm1, Psm3 and Rad21 in Schizosaccharomyces pombe). The regulatory proteins Rad61-Wapl, Pds5 and Scc3 (Wpl1,Pds5 and Psc3 respectively in S. pombe) interact with the ring via Scc1. It has been suggested that DNA capture by the cohesin complex involves the transient opening of the Smc1/Smc3 interface. The dissociation reaction involves the sub-complex Wapl/Pds5/Scc3 which likely causes the opening of the Scc1/Smc3 interface. The mechanisms by which cohesion is created and by which Wapl promotes the cohesin dissociation from chromosomes are still unknown. Among the cohesion mutants in Saccharomyces cerevisiae the thermosensitive eco1-1 mutation affects the ECO1 gene encoding an acetyl-transferase essential for cell viability and conserved from yeast to human (Eco1 « Establishment of Cohesion » in S.cerevisiae, Eso1 in S. pombe and ESCO1-2 in human) and whose substrate is Smc3. It has been shown that the acetyl-transferase counteracts the dissociation action of Wapl. A genetic screen carried out by several teams in order to find suppressors of the eco1-1 mutation has led to the identification of the genes encoding the Wapl, Pds5, Scc3 and Smc3 proteins as components of the opening mechanism of the cohesin ring. A similar screen was carried out in S. pombe in our lab to find suppressors of the thermosensitive mutation eso1-H17. This screen identified the orthologous genes to those found in the budding yeast: wpl1,pds5, psc3 and psm3 and also the gene encoding the catalytic subunit of the type 4 serine/threonine phosphatase complex (PP4) named pp4c. We have therefore carried out experiments to characterize PP4c and its regulatory subunit Psy2 which has also been found to be involved in sister-chromatid cohesion. We have likewise identified the Rad21 subunit as a PP4 substrate and identified phosphosites as potential targets of PP4. We have then screened and analyzed Rad21 phosphomutants which were able to mimic the suppressor effect of the deletion of pp4c.