<p>Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. </p><p>Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. </p><p>Several multivariate analysis tools, <i>e.g.</i> principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.</p>

A doença de Chagas, parasitose endêmica causada pelo Trypanosoma cruzi, se apresenta como grande causa de morbimortalidade, afetando cerca de 18 milhões de pessoas no continente americano e causando 21.000 mortes, a cada ano. Atualmente, estima-se que 100 milhões de pessoas estejam sob risco de contaminação nos 18 países da área endêmica da doença. A quimioterapia contra a tripanossomíase americana é constituída por apenas dois fármacos, nifurtimox e benznidazol, que não apresentam ação adequada na fase crônica da doença. Por estas razões, é premente a necessidade de novas e mais eficazes alternativas terapêuticas. A cruzaína, mais abundante cisteíno-protease do parasita, é enzima essencial em todos os estágios de modificação celular do parasita e está, também, presente no processo de invasão e modificação do sistema imune do hospedeiro. Além disso, apresenta diferenças em relação a enzimas dessa categoria no hospedeiro. Trata-se, pois, de excelente alvo bioquímico para a pesquisa de novos agentes contra o T. cruzi. Face ao exposto e tendo-se em vista a especificidade da cruzaína, considera-se de grande interesse a compreensão do mecanismo de interação de compostos com essa enzima com o intuito de planejar derivados com atividade antichagásica potencial. O presente trabalho teve o objetivo de elucidar a afinidade de diferentes classes de compostos pela enzima, na busca racional de novos tripanomicidas. Assim, pró-fármacos peptídicos recíprocos, derivados de primaquina(PQ) e de nitrofural(NF), anteriormente sintetizados, e pró-fármacos peptídicos duplos do hidroximetilnitrofural (NFOH) planejados, e cuja síntese foi estudada, foram submetidos a estudos de modelagem molecular e de docking. Os peptídios foram escolhidos com base na cisão específica pelacruzaína. Avaliou-se a interação com a enzima e elucidar o mecanismo de liberação dos fármacos e composto ativo a partir desses derivados. Os estudos indicaram que o melhor transportador a ser utilizado no planejamento de novos pró-fármacos é o dipeptídio LysArg, corroborando o que havia sido observado nos ensaios em cultura de células infectadas . com o T. cruzi. Ante a possibilidade de um segundo mecanismo de ação pela interação entre a Cys25 da cruzaína e o grupo semicarbazona, presente no NF e no NFOH, planejaram-se e sintetizaram-se análogos destes compostos, utilizando bioisosterismo clássico entre enxofre e oxigênio. Estudos de modelagem molecular e docking indicaram a participação também deste mecanismo de ação na atividade dos referidos derivados nitro-heterocíclicos. Todos os bioisósteros apresentaram ação inibitória na cruzaína IC50 entre 2,71 &#181;M e 22,83 &#181;M - evidenciando, também, este mecanismo de ação. Com o objetivo de se estudar a influência do grupamento (tio)semicarbazona presente nos derivados bioisostéricos sintetizados, realizaram-se estudos de QSAR 20 e 3D em série de compostos descritos na literatura, obtendo-se modelos robustos e com grau elevado de predição, comprovando a ação na cruzaína. Tais grupos podem ser utilizados no planejamento de novos tripanomicidas. Complementando a busca racional de novos antichagásicos potenciais, efetuou-se triagem virtual de novos ligantes utilizando o planejamento racional com base na estrutura do receptor (SBDD) a partir de modelo farmacofórico específico obtido para a cruzaína. Esse estudo proporcionou a sugestão de vinte moléculas a partir do banco de dados CHEMDIV, com possível ação inibitória da enzima. Com base nos resultados obtidos, conclui-se que os estudos realizados com pró-fármacos e análogos por meio de processos racionais de planejamento forneceram dados importantes para a pesquisa de candidatos a novos fármacos antichagásicos. / Chagas \' disease, a parasitosis caused by Trypanosoma cruzi, is an endemic disease that affects most part of Latin America. About 18 million people are infected by the parasite and around 21 thousand deaths are related to Chagas\' disease each year. Nowadays, 100 millions of people are estimated to be under the risk of infection in the 18 countries of the endemic areas. The therapeutic armamentarium available against Chagas\' disease is comprehended by only two drugs, nifurtmox and benznidazol, which are not effective in the chronic phase of the disease. Cruza in, the most abundant cysteine protease of the parasite, is essential in all stages of the cellular development of the parasite and responsible for invasion and modification of the immunologic system of the human host. Besides, it has differences relatively to those enzymes in the humans. So, it is an excellent biochemical target for searching new agents against T. cruzi. This said and in the view of cruzain specificity, understanding the mechanism of interaction of compounds with this enzyme is considered very interesting in order to design derivatives with potential anti-Chagas\'disease. The present work had the objective of elucidating the affinity of different classes \'of compounds to the enzyme, in the rational search for new trypanomicides. So, mutual peptide prodrugs, derived from primaquine (PQ) and nitrofurazone (NF), previously synthesized, and designed double peptide prodrugs of hydroxymethylnitrofurazone (NFOH), which synthesis has been studied, were submitted to molecular modeling and docking studies. The peptides were chosen based on specific cleavage by cruzain. The interaction with the enzyme and the drug as well as the active compound release mechanism from those derivatives were evaluated. The studies have indicated the dipeptide LysArg as the best carrier to be used in the design of new prodrugs, corroborating what had been earlier observed in the tests of T. cruzi infected cell culture. Based on the possibility of a second mechanism of action through the interaction between cruzain Cys 25 and the semicarbazone group, found in NF and NFOH, analogs of this c/ass of compounds were designed, and synthesized, using classic bioisosterism between sultur and oxygen. Molecular modeling and docking studies have indicated also the participation of this mechanism in the activity of the nitro-heterocyclic compounds referred. All bioisosters showed to inhibit cruzain IC50 between 2.71 &#181;M and 22.83 &#181;M -- also evidencing this mechanism of action. With the purpose of studying the influence of (thio)semicarbazone group present in the synthesized bioisoster derivatives, 20 and 3D QSAR have been developed for a series of compounds reported in the literature, leading to robust and high- preditive leveI models, confirming their action in cruzaine. Those groups might be used in the design of new trypanomicides. Complementing the rational search for new antichagasic compounds, a virtual screening of new ligands, using the structure-based drug design (SBDD) was developed based on a specific pharmacophore model obtained for cruzain. This study have provided the suggestion of twenty compounds from the CHEMDIV data bank with possible inhibitory activity in cruzain. Based on the results obtained, the conclusion is that the studies herein developed with prodrugs and analogs through rational drug design lead to important data towards the research of new candidates as new antichagasic drugs.

A malária ainda constitui grave problema de saúde pública, estando presente principalmente em países em desenvolvimento. Acredita-se que 40% da população mundial vivam em áreas endêmicas para esta parasitose. No Brasil, cerca de 600 mil novos casos aparecem a cada ano, merecendo realce a Amazônia, que corresponde a 99% dos casos brasileiros. Dentre os agentes etiológicos, o Plasmodium falciparum suscita atenção especial, pois é o responsável pela malária grave. A artemisinina é agente antimalárico cujo emprego encontra-se principalmente para cepas resistentes. Embora este fármaco seja o único antimalárico cuja resistência dos plasmódios não possui relevância clínica, problemas de solubilidade comprometem sua eficácia clínica e fazem com que seja utilizado sempre em associação a antimaláricos com maior meia-vida plasmática. Com o objetivo de aumentar a biodisponibilidade do antimalárico, conferindo-lhe maior hidrossolubilidade e ante ao emprego de quitosana como transportador com vistas, entre outros, a esse objetivo, planejaram-se pró-fármacos de quitosanas modificadas e artemisinina. Inicialmente, obtiveram-se quatro derivados hidrofílicos da quitosana: N-carboxibutilquitosana, N-carboximetilquitosana, N,O-carboximetilquitosana e N-succinilquitosana. A artemisinina foi empregada na forma reduzida, a diidroartemisinina, sintetizada a partir do fármaco na presença de boroidreto de sódio em várias proporções relativas ao fármaco. Procedeu-se, então, à condensação entre a diidroartemisinina e os derivados hidrofílicos obtidos por meio de quatro métodos distintos. Prepararam-se, também, microesferas para incorporar e ligar covalentemente a diidroartemisinina à quitosana. O fármaco flurbiprofeno foi utilizado como fármaco lipofílico modelo de reação. Os resultados obtidos indicam a formação de microesferas de pró-fármaco de quitosana com artemisinina, utilizando genipina como reticulante, empregando-se emulsão múltipla O/A/O juntamente com as microesferas. Os derivados obtidos, uma vez confirmada inequivocamente a estrutura, serão submetidos a ensaios de dissolução intrínseca e de atividade em malária experimental por P. berghei, em animais. / Malaria is still a serious problem of public health, mostly in developing countries. About 40% of the world population live in malaria endemic areas. In Brazil, about 600 thousands new cases appear every year, mainly in Amazonia, in which 99% of the cases have been registered. Among the etiologic agents, Plasmodium falciparum deserves special attention, as it is responsible for the severe malaria. Artemisinin is an antimalarial agent mostly used for resistant strains. Although this drug is the only antimalarial whose resistance of plasmodio has not clinical relevance, solubility problems compromise its clinical effectiveness. This is the reason why it has been used mainly in combination with other antimalarial drugs with higher t1/2.. With the goal of increasing the drug biovailability, rendering it with higher hydrosolubility, and since modified chitosans have been used as carriers to impart this characteristic, among others, to the drugs, artemisinin prodrugs with chitosan were designed. Initially, four hydrophilic derivatives were obtained: N-carboxybutylchitosan, N-carboxymetylchitosan, N,O-carboxymetylchitosan and N-succinylchitosan. Artemisinin was utilized in its reduced form, dihydroartemisinin, synthesized from the drug in the presence of sodium borohydride in several equivalents related to the drug. The condensation between dihydroartemisinin and hydrophilic chitosan derivatives was carried out by four distinct methods. In addition, microspheres to incorporate artemisinin and to attach dihydroartemisinin to the chitosan were prepared. Flurbiprofen was used as lipophilic drug reaction model. The results indicated the dihydroartemisinin prodrug microspheres, using genipin as crosslinking agent by multiple emulsion method O/A/O, were obtained. Once unequivocally confirmed their structure, these derivatives will be submitted to intrinsic dissolution test and to biological activity determination in experimental malaria with Plasmodium berghei.

A malária ainda constitui grave problema de saúde pública, estando presente principalmente em países em desenvolvimento. Acredita-se que 40% da população mundial vivam em áreas endêmicas para esta parasitose. No Brasil, cerca de 600 mil novos casos aparecem a cada ano, merecendo realce a Amazônia, que corresponde a 99% dos casos brasileiros. Dentre os agentes etiológicos, o Plasmodium falciparum suscita atenção especial, pois é o responsável pela malária grave. A artemisinina é agente antimalárico cujo emprego encontra-se principalmente para cepas resistentes. Embora este fármaco seja o único antimalárico cuja resistência dos plasmódios não possui relevância clínica, problemas de solubilidade comprometem sua eficácia clínica e fazem com que seja utilizado sempre em associação a antimaláricos com maior meia-vida plasmática. Com o objetivo de aumentar a biodisponibilidade do antimalárico, conferindo-lhe maior hidrossolubilidade e ante ao emprego de quitosana como transportador com vistas, entre outros, a esse objetivo, planejaram-se pró-fármacos de quitosanas modificadas e artemisinina. Inicialmente, obtiveram-se quatro derivados hidrofílicos da quitosana: N-carboxibutilquitosana, N-carboximetilquitosana, N,O-carboximetilquitosana e N-succinilquitosana. A artemisinina foi empregada na forma reduzida, a diidroartemisinina, sintetizada a partir do fármaco na presença de boroidreto de sódio em várias proporções relativas ao fármaco. Procedeu-se, então, à condensação entre a diidroartemisinina e os derivados hidrofílicos obtidos por meio de quatro métodos distintos. Prepararam-se, também, microesferas para incorporar e ligar covalentemente a diidroartemisinina à quitosana. O fármaco flurbiprofeno foi utilizado como fármaco lipofílico modelo de reação. Os resultados obtidos indicam a formação de microesferas de pró-fármaco de quitosana com artemisinina, utilizando genipina como reticulante, empregando-se emulsão múltipla O/A/O juntamente com as microesferas. Os derivados obtidos, uma vez confirmada inequivocamente a estrutura, serão submetidos a ensaios de dissolução intrínseca e de atividade em malária experimental por P. berghei, em animais. / Malaria is still a serious problem of public health, mostly in developing countries. About 40% of the world population live in malaria endemic areas. In Brazil, about 600 thousands new cases appear every year, mainly in Amazonia, in which 99% of the cases have been registered. Among the etiologic agents, Plasmodium falciparum deserves special attention, as it is responsible for the severe malaria. Artemisinin is an antimalarial agent mostly used for resistant strains. Although this drug is the only antimalarial whose resistance of plasmodio has not clinical relevance, solubility problems compromise its clinical effectiveness. This is the reason why it has been used mainly in combination with other antimalarial drugs with higher t1/2.. With the goal of increasing the drug biovailability, rendering it with higher hydrosolubility, and since modified chitosans have been used as carriers to impart this characteristic, among others, to the drugs, artemisinin prodrugs with chitosan were designed. Initially, four hydrophilic derivatives were obtained: N-carboxybutylchitosan, N-carboxymetylchitosan, N,O-carboxymetylchitosan and N-succinylchitosan. Artemisinin was utilized in its reduced form, dihydroartemisinin, synthesized from the drug in the presence of sodium borohydride in several equivalents related to the drug. The condensation between dihydroartemisinin and hydrophilic chitosan derivatives was carried out by four distinct methods. In addition, microspheres to incorporate artemisinin and to attach dihydroartemisinin to the chitosan were prepared. Flurbiprofen was used as lipophilic drug reaction model. The results indicated the dihydroartemisinin prodrug microspheres, using genipin as crosslinking agent by multiple emulsion method O/A/O, were obtained. Once unequivocally confirmed their structure, these derivatives will be submitted to intrinsic dissolution test and to biological activity determination in experimental malaria with Plasmodium berghei.

Matrix metalloproteinases (MMPs) play a significant role in degrading the extracellular matrix in cancer development and metastasis. Overexpression of matrix metalloproteinases in tumour tissues relative to normal tissues has been exploited as a target for peptide-based therapeutics, to improve therapeutic index of currently used agents. The stability of MMP-activated prodrugs in normal tissue or organs is a significant challenge for their success in the clinic. In an in vitro study, the stability of twenty six prodrugs was studied in mouse liver, kidney, lung and tumour homogenates using HPLC and LC/MS. Selected agents were studied in vivo. Each prodrug has a characteristic amino acid sequence with dominant FITC N-terminal end cap. All prodrugs were conjugated to a colchicine derivative (ICT 2552) which is a vascular disrupting agent causing tumour vasculature shutdown and consequently, tumour necrosis. ICT 3146, ICT 3019, ICT 3120 and ICT 3115 prodrugs showed significant stability in normal tissues and considerable activation in certain tumour tissues compared to the lead compound ICT 2588. Also, the selectivity of promising prodrugs to the MMP family was confirmed by using leupeptin (serine, cysteine and threonine protease inhibitor), pepstatin A (aspartate protease inhibitor), phosphoramidon (nepralysin inhibitor), ilomastat (metalloproteinase inhibitor) and BML-P115 (matrix metalloproteinase inhibitor). Moreover, members of the MMP family responsible for cleaving the selected prodrugs were identified using recombinant MMP enzymes. Furthermore, a LC/MS-MS method was developed to specifically detect and quantify MMP-16 protein expression in H460 tumour. MMP- 16 was responsible for the cleavage of ICT 3146 and ICT 3115. Therefore, MMPactivated prodrugs could be a useful therapeutic approach to avoid off-site toxicities of currently used anti-tumour agents.

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinicallyuseful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient.

Doenças cardiovasculares podem ocasionar manifestações clínicas graves como infarto agudo do miocárdio e acidentes vasculares trombóticos, constituindo a principal causa de morte no mundo, fato esse que desperta grande interesse da indústria farmacêutica. As causas normalmente estão relacionadas à elevação dos níveis de colesterol e à agregação plaquetária, que acarretam eventos vaso-oclusivos. Entre as alternativas terapêuticas para o controle e prevenção das doenças cardiovasculares podem-se destacar os inibidores da 3-hidroxi-3-metilglutaril coenzima-A redutase (HMG-CoA redutase), popularmente conhecidos como estatinas. A rosuvastatina merece destaque nessa classe de fármacos, devido à maior seletividade e potência na redução dos níveis de colesterol LDL. O ácido acetilsalicílico, antiinflamatório não-esteróide, também representa uma importante alternativa terapêutica para prevenção de doenças cardiovasculares, devido à sua ampla aceitação como inibidor da agregação plaquetária. Considerando seus mecanismos de ação, estatinas e ácido acetilsalicílico podem ser usados em conjunto para a prevenção de doenças cardiovasculares. Face ao exposto e tendo-se em vista a importância dos dendrímeros como transportadores de fármacos na latenciação, o presente trabalho teve como objetivo desenvolver o pró-fármaco dendrimérico potencialmente ativo em doenças cardiovasculares contendo rosuvastatina e ácido acetisalicílico. Diversas metodologias de síntese foram realizadas na tentativa de obtenção do pró-fármaco dendrimérico composto por mio-inositol ou etilenoglicol como foco central, ácido L(-)-málico e etilenoglicol como espaçantes e rosuvastatina e ácido acetilsalicílico como compostos bioativos. Parte dos intermediários propostos foi sintetizada e purificada com sucesso. As maiores dificuldades encontradas foram a purificação dos compostos e a hidrólise seletiva da proteção do ácido málico protegido. Adicionalmente, realizaram-se estudos computacionais para prever a liberação dos fármacos do pró-fármaco dendrimérico. / Cardiovascular diseases can lead to several clinical manifestations such as myocardial infarction and stroke. Those diseases represent the main cause of death globally and this fact triggers a great interest from the pharmaceutical industries. The causes are usually related to high cholesterol levels and platelet aggregation, which are responsible for the vaso-occlusive events. Among the available drug therapy for control and prevention of cardiovascular diseases, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), popularly known as statins, can be highlighted. Rosuvastatin deserves mention in this class of drugs due to its greater selectivity and potency in reducing the levels of LDL cholesterol. Aspirin, a nonsteroidal anti-inflammatory, also represents an important drug therapy for treatment and prevention of cardiovascular diseases, due to its widespread acceptance as a platelet aggregation inhibitor. Considering their mechanism of action, aspirin and statin can be used in association for prevention of cardiovascular diseases. This said and taken into account that dendrimers are important as carriers in prodrug design the purpose of this work was the synthesis of dendrimer prodrugs potentially active in cardiovascular diseases containing aspirin and rosuvastatin. Several synthetic methods have been used with the aim to synthesizing the dendrimer produgs composed of myo-inositol or ethyleneglycol as core, L-(-)-malic acid and ethyleneglycol as spacer groups and rosuvastatin and aspirin as bioactive compounds. Some of the proposed intermediates was synthesized and purified successfully. The main difficulties were purification of compounds and selectivy desprotection of protected malic acid. Additionally, computational studies were performed in order to predict the release of those drugs from dendrimer prodrugs

Objective: To review the place in therapy of isavuconazole, the active metabolite of isavuconazonium sulfate, via a review of the available literature on drug chemistry, spectrum of activity, pharmacokinetic/pharmacodynamic profile and trials assessing clinical efficacy and safety. Methods: Relevant data, original research articles and reviews, were gathered primarily through the use of a PubMed database search. The search was conducted without date restrictions in order to collect both historical and recent data regarding isavuconazole. Key findings: Isavuconazole is a triazole currently approved not only for use in invasive aspergillosis and mucormycosis but also has demonstrable activity against Candida species and other common fungal pathogens. This drug has features which make it more clinically appealing compared to other azoles with similar indications. In specific, isavuconazole does not require a cyclodextrin vehicle due to its water solubility, and at present, does not require therapeutic drug monitoring. Moreover, isavuconazole has displayed improved safety and tolerability compared to voriconazole. Available data from Phase III clinical trials shows isavuconazole to be a possible therapeutic option to currently available therapies for which it is approved; however, clinical conclusions should be reserved until results have been published and more data from clinical use is reported. Conclusions: Isavuconazole is a new triazole with broad‐spectrum antifungal activity including invasive aspergillosis and mucormycosis.

En raison d’une abondante matrice extracellulaire chondrogénique, peu vascularisée et hypoxique, le chondrosarcome, cancer du cartilage, est une tumeur chimio et radiorésistante. Une stratégie bispécifique s’appuyant sur deux caractéristiques spécifiques du chondrosarcome, une forte densité de charges négatives, ainsi qu’une hypoxie chronique sévère de la matrice extracellulaire, est actuellement développée au laboratoire et consiste à fonctionnaliser une prodrogue activée en hypoxie avec un ammonium quaternaire (AQ). Sur la base de résultats précliniques, une prodrogue, ICF05016, a été identifiée et fait actuellement l’objet d’une étude d’optimisation.Ce projet vise à identifier des relations structure-activité dans une série de moutardes phosphorodiamidates vectorisées par une fonction AQ. Dans une première partie, nous nous sommes intéressés à faire varier la nature des substituants de la fonction ammonium quaternaire ainsi que la longueur du bras espaceur séparant cette dernière de la moutarde. Ainsi, 27 dérivés ont été synthétisés par une séquence commune multi-étapes impliquant la phosphorylation d’un dérivé 2-nitro-5-hydroxyméthylimidazole.Un screening par résonance plasmonique de surface a permis de mettre en évidence 12 composés plus affins envers l’aggrécane que le composé de référence ICF05016. Cette étude a révélé une dépendance importante entre l’affinité à l’aggrécane et la nature de l’ammonium quaternaire, les composés benzyléspossédant tous des affinités supérieures. In vitrosur cultures cellulaires HEMC-SS, les composés ont affichés des ratios de cytotoxicité normoxie vs hypoxie (HCR)supérieurs à celui du lead ICF05016, et notamment le composé benzylé31f.Avec une constante de dissociation à l’aggrécane de 2,10 µM et un différentiel de cytotoxicité normoxie/hypoxie équivalent à celui de l’évosfosfamide en étude clinique, la composé 31f a été évalué en termes de stabilité et de clivage en conditions réductrices, mimant l’hypoxie tumorale. Démontrant une excellente stabilité sur 24 heures dans un tampon phosphate, le composé 31f s’est révélé être réduit par réduction chimique ou enzymatique via une nitroréductase, et ce avec une cinétique rapide. Ce dernier a été ainsi identifié au terme de l’évaluation in vitro et de cet axe de recherche comme un nouveau « lead ».Dans une seconde partie, nous avons choisi de moduler l’halogène et la position des bras alkylants portés par la moutarde phosphorodiamidate. Compte tenu de l’instabilité des composés de la série bromée, par rapport à la série chlorée, les analogues bromés des composés ICF05016 et 31f n’ont pu faire l’objet d’une évaluation in vitro. Pour les isomères de type moutarde ifosfamide, l’instabilité des précurseurs phosphoramidiques et les difficultés de synthèse inhérentes à l’introduction d’une fonction amine secondaire sur un chlorure de phosphoryle préalablement fonctionnalisé, nous ont contraints à réorienter nos travaux vers des structures issues d’une étape de chimie de click, permettant d’introduire le bras vecteur portant la fonction amine tertiaire indépendamment de l’étape de phosphorylation. Cette séquence réactionnelle pourrait être étendue à la synthèse de nouveaux analogues du composé 31f, identifié lors de ces travaux. / Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. A dual targeted therapy leveraging specific chondrosarcoma hallmarks, an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia,was investigatedby conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions. Based on preclinical results, animidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study.This project was designed to identify structure-activity relationships in a series of QA-targeted phosphorodiamidate mustard conjugates. In a first part, a series of 27 conjugates, with different QA function and length of the alkyl linker, was synthesizedvia a common multi-step sequence involvingphosphorylation of a key 2-nitroimidazole alcohol intermediate. Then, biomolecular interactions between these QA derivatives and aggrecan were assessed using thesurface plasmon resonance technology. This screeningrevealed that the affinity depends more on the natureof the QA function, rather than on the linker length. The most promising results were obtainedwith QA bearing a benzyle group. Twelves compounds were then evaluated in terms of hypoxia selective cytotoxicityonthe HEMC-SS cell line. For all prodrugs, an overall improvement in hypoxic selectivity compared with the ICF05016 was obtained and the positive impact of thebenzyle QA function was again highlighted. With a dissociation constant of 2.10 µM in the SPR experiment and an attractive hypoxic selectivity, compound 31fwas further selected for a stability and reductive activation study. Activation of the prodrug and phosphoramide mustard release under reductive chemical conditions, and nitroreductase-based activation were demonstrated. From this study, compound 31f emerged as the most effective PG-targeted HAPs. In a second part, the nature of the halogen and the position of the alkylating arms carried by the phosphorodiamidic mustard were modified. Given the high instability of the final compounds of the brominated series, compared to the chlorinated series, brominated analogs of the compounds ICF05016 and 31fcould not be obtained with sufficient purity to be evaluated in vitro.For ifosfamide-like isomers, the design of the prodrugs was modified due to the instability of the phosphoramidic precursors or difficulties of introducing a secondary amine function on a phosphoryl chloride previously functionalized.A click chemistry approach was developed to tether the spacer arm carrying the tertiary amine function independently of the phosphorylation step and could be extended to the synthesis of triazoleanalogues of the new lead 31f.

<p>A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in <i>E. coli</i>. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.</p>