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Les Rhabdomyosarcomes (RMS) sont des sarcomes qui touchent préférentiellement les enfants et les adolescents. Les RMS sont à l'origine de nombreuses métastases qui sont responsables d'une réduction importante de l'espérance de vie du malade. Une meilleure compréhension des mécanismes sous-tendant la migration et l'invasion des RMS pourrait orienter vers de nouvelles thérapies visant à enrayer le développement de métastases. La dissémination métastatique fait intervenir de nombreuses protéases dont la µ- et la m-calpaïne, cystéine-protéases, constituant avec leur inhibiteur endogène, la calpastatine, le système protéolytique neutre calcium-dépendant. Dans différents travaux, l'activité de ces calpaïnes a été montrée comme dérégulée, notamment dans le cancer du rein, de la peau ou encore les adénocarcinomes colorectaux. Une connaissance approfondie de l'impact de la dérégulation de l'activité des calpaïnes sur la dissémination métastatique pourrait en faire des cibles thérapeutiques de choix. L’étude de l’activité, de l’expression et de la localisation des différents composants du système protéolytique neutre calcium-dépendant a permis de mettre en évidence une activité dérégulée des calpaïnes dans les RMS. Cette forte activité serait due à une expression très faible de la calpastatine. L’analyse comparative des caractéristiques adhésives et cinétiques des RMS par rapport aux cellules témoins, des myoblastes humains (LHCN-M2) montre un faible taux d’adhésion associé à une vitesse de migration élevée des RMS. L’activité calpaïne présente une corrélation linéaire positive avec la vitesse de migration ; les calpaïnes se présentent donc comme marqueur de l’agressivité tumorale. L’inhibition des calpaïnes par la calpeptine réduit fortement cette vitesse. Le cytosquelette des RMS est désorganisé et ne présente pas, contrairement à celui des cellules non-tumorales, de fibres de stress. À ce niveau, les études pour tenter de discriminer le rôle de la µ-calpaïne et de la m-calpaïne, utilisant des oligonucléotides antisens, montrent que dans les LHCN-M2, la µ-calpaïne jouerait un rôle prépondérant dans la régulation de l’alpha-actine en régulant de manière négative son expression. Quant à la béta-actine, elle serait régulée, aussi de manière négative, mais, uniquement par la m-calpaïne. Dans les ARMS, la µ-calpaïne et la m-calpaïne jouent un rôle similaire en stimulant l’expression des deux isoformes. De plus, le pouvoir invasif important des RMS est fortement diminué lorsque l’activité calpaïne est inhibée. L’implication des calpaïnes tant au niveau de l’adhésion, de la migration que de l’invasion des RMS font de ces dernières une cible d’étude importante pour tenter de contrecarrer le développement de métastases. / Rhabdomyosarcoma (RMS) are a soft-tissue sarcoma commonly encountered in childhood and adolescence. RMS cells can acquire invasive behaviour and can form metastases which decrease less than 20% the patients healing. The comprehension of mechanisms that regulate cancer cells migration and invasion may be a key for development of new therapies for limiting metastasis. The metastatic dissemination implicates lots of proteases of which µ-calpain and m-calpain. Calpain activity is Ca2+-dependent and is principally regulated by calpastatin, its specific endogenous inhibitor. The deregulation of calpains has been involved in tumour invasion and metastasis in several different cell types. Then, calpains would be a good target for development of novel therapies to control metastasis. Study of calpain activity, expression, and localisation underline the deregulation of calpain activity in RMS. This high activity may be due to weak expression of calpastatin. The comparative analysis of adhesive and kinetical characteristics of RMS cells, compared to human myoblasts, LHCN-M2 cells, show a weak adhesiveness and an important migration velocity in RMS cells. The calpain activity presents a positive linear correlation with the migration velocity; so, calpain may be considered as marker of tumoral aggressiveness. The inhibition of calpain by calpeptin reduces significantly the migration velocity. The cytoskeleton RMS cells is disorganised and does not present stress fibers, contrary to LHCN-M2 cells. At this level, discrimination of µ- and m-calpain role, using antisens oligonucleotides, shows that in LHCN-M2 myoblasts, µ-calpain may negatively regulate the alpha-actin expression and that béta-actin may be positively regulated by the m-calpain. In ARMS cells, both µ- and m-calpain positively regulate alpha- and béta-actin. Moreover, the invasive behaviour of RMS cells is importantly decreased when calpains are inhibited. In summery, calpains may implicate in the anarchic adhesion, migration and increase of invasion. In this way, targeting calpain activity may represent a good strategy for limiting development of RMS tumour as well as their metastatic behaviour.

Duchene muscular dystrophy (DMD) is a lethal X-linked disease that afflicts approximately 1 in 3500 newborn males. Boys with DMD will become progressively weaker causing wheelchair dependence by their early teens and death by their mid to late twenties. Currently there is no cure for DMD, the exact mechanism of disease action remains elusive, and treatments to improve quality of life are limited. Two areas of DMD research that could begin to fill this void and provide simple, cost effective therapy aimed to improve quality of life are neutriceutical and exercise therapies. We hypothesized that leucine, a branched chain amino acid (BCAA) with anabolic properties, given to sedentary and exercised x-linked dystrophic mice (mdx) over 4 weeks would improve skeletal muscle function and decrease markers of skeletal muscle degradation. In sedentary mdx mice, leucine improved tetanic extensor digitorum longus (EDL) stress (p < 0.05), gastrocnemius mammalian target or rapamycin (mTOR) phosphorylation (p < 0.05), while decreasing the rate of real-time calpain activity in flexor digitorum brevis (FDB) fibers (p < 0.05) compared to sedentary mice given no leucine. In exercised mdx mice, leucine improved total running distance over the 4 week testing period by 40% (p < 0.02) and increased EDL stress at every frequency recorded (p < 0.05). Our data lead us to the conclusion that the BCAA leucine can increase EDL muscle stress in dystrophic animals, and that the effects of leucine treatment are enhanced when leucine supplementation is combined with exercise. Leucine supplementation should be explored further and in higher order species of muscular dystrophy to determine if its use could provide clinical improvements in DMD patients. / Ph. D.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF-hand motif characteristic of the classical calpains, calpain 1 and 2, but retains catalytic and Ca2+ binding non EF domains. Tra-3, an ortholog of CAPN5, is involved in necrotic cell death in C.elegans; although specific role of CAPN5 has not been investigated in the mammalian CNS. I compared relative mRNA levels of calpains in rat CNS, which revealed that CAPN5 is the second most highly expressed calpain. We examined relative levels of CAPN5 from late embryonic day 18 to postnatal day 90 and found lower mRNA but higher protein levels during CNS development. Using X –gal staining in Capn5 +/- mice, immunostaining of rat brain sections and SH-SY5Y cells, and subcellular fractionation of rat brain cortex, we found that CAPN5 is a non-cytoplasmic calpain localized in the nucleus and enriched in synaptic mitochondria. Proteinase K treatment of mitochondria and mitoplasts from B35 rat neuroblastoma cells and rat synaptic mitochondria revealed CAPN5 was localized on the inner mitochondrial membrane and released from mitochondria on membrane permeabilization with alamethicin. We used immunolabelling, confocal imaging, nuclear subfractionation and transient transfections to evaluate the subnuclear localization of CAPN5. CAPN5 was detected in punctate domains and associated with promyelocytic leukemia (PML) protein, a tumor suppressor protein. We further demonstrated that CAPN5 carries a nonconventional bipartite nuclear localization signal. Together, these findings demonstrate that CAPN5 is a non-cytosolic calpain, abundant in the CNS and localized to the mitochondria inner membrane and nuclear PML bodies.

Le cancer colorectal est un cancer majeur en termes de fréquence et de mortalité. Il s’agit de la deuxième cause de décès par cancer, avec 17 500 décès en France en 2011. Le traitement des stades avancés repose sur différentes molécules anti-cancéreuses telles que l’oxaliplatine. Cependant le développement de résistances entraine des échecs thérapeutiques expliquant le faible taux de survie observé. Il est donc crucial d’identifier les mécanismes de résistance et ses acteurs et de découvrir de nouvelles pistes de traitements.Dans un premier temps, nous nous sommes donc intéressés aux rôles joués par les calpaïnes et NOX1 dans le développement de la résistance à l’oxaliplatine en étudiant des cellules tumorales colorectales résistantes à cette drogue. Ce qui nous a permis d'identifier une voie de signalisation impliquée dans la résistance à cette chimiothérapie.Dans un second temps nous nous sommes intéressés à étudier la réversion de cette résistance à l’oxaliplatine. En criblant différentes chimiothérapies ce qui a permis de mettre en évidence une inversion du statut résistant/sensible dans nos cellules sélectionnées.La première partie de nos données met en évidence de nouvelles régulations de Nox1 qui diffèrent en fonction de la sensibilité des cellules à l’oxaliplatine. Nos résultats montrent aussi que p38 MAPK pourrait être une cible thérapeutique de choix. Dans la deuxième partie nous avons identifié un nouveau traitement permettant l'induction de l'apoptose dans nos cellules résistantes. Ainsi la gemcitabine pourrait être une solution pour traiter les patients ne répondant pas ou plus aux protocoles basés sur l’oxaliplatine / Colorectal cancer is a major cancer in terms of frequency and mortality. This is the second leading cause of cancer death, with 17,500 deaths in France in 2011. The treatment of advanced stages is based on different chemotherapeuties including oxaliplatin. However, the development of resistance leads to therapeutic failures explaining the low survival rate. It is therefore crucial to identify the mechanisms of resistance and the actors involved and to discover new therapeutic approaches. We first investigated the role played by calpains and NOX1 in the development of resistance to oxaliplatin, studying oxaliplatin-resistant colorectal tumor cells. This allowed us to identify a signaling pathway involved in resistance to this chemotherapy.Secondly, we have studied the reversion of this resistance to oxaliplatin. A screening of different chemotherapies revealed a reversal of the resistant / sensitive status in our selected cells In the first part, our data highlight novel Nox1 regulations which differ according to the sensitivity of the cells to oxaliplatin. Our results also show that p38 MAPK could be a therapeutic target for treating colorectal cancers resistant to oxaliplatin. In the second part, we have identified a new treatment to induce apoptosis in our resistant cells. Indeed, gemcitabine may be a solution to treat patients who do not respond to oxaliplatin-based protocols.

La sarcopénie ou perte involontaire progressive de la masse musculaire chez le sujet âgé s’accompagne de l’altération de nombreux phénomènes physiologiques comparables à ceux observés chez les myopathes. Le processus de régénération musculaire est très ralenti, les activités protéolytiques intracellulaires sont modifiées et de nombreuses fonctions cellulaires sont perturbées en raison d’un stress oxydatif incontrôlé. L’intervention des calpaïnes, protéases neutres calcium-dépendantes, dans les processus associés au développement, à la régénération et à l’intégrité du tissu musculaire est incontestable. Les calpaïnes apparaissent, en effet, comme des acteurs clefs des voies de transductions liées à la myogenèse, la prolifération et la survie cellulaire. Toutefois aucune étude permettant d’établir la relation vieillissement du tissu musculaire- activité calpaïne n’a été entreprise à ce jour. Le projet a donc pour but principal d’inventorier les signaux pro-sarcopéniques interagissant avec les calpaïnes et d’établir leurs relations avec la fonctionnalité des cellules satellites, le stress oxydant et l’apoptose. Nous avons mis en évidence une augmentation de l’expression/activité des calpaïnes durant le vieillissement musculaire chez le rat et identifié des partenaires des calpaïnes impliqués dans des fonctions physiologiques altérées durant la sarcopénie: homéostasie calcique, activité contractile, production d’ATP, régénération musculaire. Nous avons également montré que l’induction d’un stress oxydant entraîne l’activation des calpaïnes au cours de la prolifération des cellules satellites de façon corrélée à une augmentation de l’apoptose. D’une manière intéressante, un traitement préventif par un antioxydant naturel d’écorce de pin (Oligopin®) est capable de prévenir à la fois l’apoptose et l’activation des calpaïnes. L’ensemble de ces résultats suggère que le stress oxydant associé au vieillissement induirait des mécanismes calpaïno-dépendants responsables de l’altération de processus essentiels à la fonction musculaire. / Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern. Although sarcopenia is well documented, the molecular mechanisms of this condition still remain unclear. The calcium-dependent proteolytic system is composed of calcium dependent cystein-proteases named calpains. Calpains are involved in a large number of physiological processes such as muscle growth and differentiation, and pathological conditions such as muscular dystrophies. The aim of this study was to determine the involvement of the proteolytic system in the phenotype associated with sarcopenia by identify the key proteins (substrates or regulators) interacting with calpains during muscle aging and identify pro-sarcopenic signals after oxidative stress induction in satellite cells. Muscle aging was correlated with the up-regulation of calpain activity. Ryanodine receptor 1, ATP synthase subunit alpha and alpha actinin 3 appear as key partners of calpains during muscle aging. Such interactions suggest an implication of calpains in many processes altered during aging including cytoskeletal disorganisation, regulation of calcium homeostasis and mitochondrial dysfunction. Furthermore, oxidative stress induction led to an increase in the activity of calpains correlated to an increase in apoptosis of proliferating satellite cells. In a very interesting way, a preventive treatment with a commercial antioxidant (Oligopin®) prevented these effects. All these data suggest that oxidative stress coupled observed during muscle aging could lead to calpaïno-dependent mechanisms responsible for apoptosis and muscle dysorganisation.

This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.

This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.