<p>Lectin-functionalized, Poly [oligo(ethylene glycol) methyl ether methacrylate] (<em>POEGMA</em>) loaded with 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) and poly(ethylene glycol)–block-poly(D,L-lactic-co-glycolic acid) (PEG-PLGA) nanoparticles loaded with haloperidol were prepared with narrow size distributions and sizes < 135 nm. The microgels and nanoparticles exhibited high <em>Solanum tuberosum </em>lectin (STL) conjugation efficiencies, encapsulation efficiencies, and drug loading capacities. The <em>in vitro</em> release of PAOPA and haloperidol was slow in physiological conditions over 96 hours, demonstrating minimal drug leakage and the potential for efficient drug transport to the targeted brain tissue. POAPA, POEGMA and the STL-functionalized POEGMA microgels were found to be non-toxic in both cell lines, indicating that they would not be toxic when administered intranasally or when they reach the brain. The nasal epithelial cell uptake of rhodamine-labelled microgels was higher in cells when the STL-functionalization was present. All haloperidol-loaded nanoparticle formulations were found to be highly effective at inducing catalepsy, while intranasal administration of STL-functionalized nanoparticles using the intranasal spray device increased the brain tissue haloperidol concentrations by 2-3.5 fold compared to STL-functionalized particles administered intranasally with a pipette. For the first time, brain tissue concentrations of rhodamine-labelled microgels confirmed that microgels are capable of passing the blood-brain barrier and that this uptake is size dependent. These formulations demonstrate promise in the reduction of the drug dose necessary to produce a therapeutic effect with antipsychotic drugs for the treatment of schizophrenia using a non-invasive route of administration.</p> / Master of Science (MSc)

Transition metal bis(thiosemicarbazone) complexes have been of great interest in the last five decades. One of the most striking features of these complexes is that they possess a wide range of biological properties including antimalarial, antibacterial and anticancer activity. Zinc and copper bis(thiosemicarbazone) complexes have recently attracted attention due to their intracellular fluorescence and anticancer activity, respectively. The present work “Targeting Cancer Cells and Live Cell Imaging Using Bis(thiosemicarbazone) Complexes of Copper and Zinc” is an effort to target cancer cells using folic acid or biotin linked anticancer active copper bis(thiosemicarbazone) complexes. Interestingly, bis(thiosemicarbazone) ligands form zinc complexes that could be used to image cancer cells and one of the ligands could be used for imaging zinc in the cells. Chapter 1, provides a brief introduction to metal complexes in medicine. Different classes of metallodrugs and their mechanism of action are listed. A short discussion on different types of diagnostic drugs and transition metal complexes possessing anticancer activity is presented. An overview of the strategies available to target cancer cells is included. Furthermore, the use of thiosemicarbazone compounds for anticancer activity is reviewed in detail. Recent examples of bis(thiosemicarbazone) compounds in medicinal studies is briefly mentioned. This section ends with the scope of the present work which involves bis(thiosemicarbazone) complexes of zinc and copper. Chapter 2, “Zinc bis(thiosemicarbazone) complexes for live cell imaging and anticancer activity” deals with the synthesis and characterization of a series of mononuclear and binuclear zinc bis(thiosemicarbazone) complexes by varying substituents at the diketone moiety or at the thiosemicarbazide fragment of the ligand. The crystal structures of mononuclear ligand benzil-bis(4-pyrrolidine-3-thiosemicarbazone) (BTSCH2), zinc glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) [Zn(GTSC)]3 and [Zn(BTSC)(DMSO)] complexes were determined using single-crystal X-ray crystallography. Here, the mononuclear zinc complexes were utilized as live cell imaging agents whereas binuclear zinc complexes proved to be anticancer agents. Among the many mononuclear complexes prepared, the trimeric zinc complex derived from glyoxal- bis(4-methyl-4-phenyl-3¬thiosemicarbazone) was found to be the most fluorescent complex owing to its unique structure. This permitted live cell imaging in a number of cancer cell lines. In comparison with the well studied zinc biacetyl-bis(4-methyl-3-thiosemicarbazone) Zn(ATSM) complex, which was used as a reference, [Zn(GTSC)]3 had a 2.5 fold higher fluorescence quantum yield in DMSO. The cellular fluorescence was measured in collaboration with Prof. K.Somasundaram’s laboratory at MCBL using flow cytometry. It was observed that [Zn(GTSC)]3 had 3 to 12 fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy studies established that [Zn(GTSC)]3 localizes in the nucleus of human breast cancer MCF-7 and MDA-MB-231 cells within 30 minutes of addition. Moreover, [Zn(GTSC)]3 showed relatively less cytotoxicity compared to the Zn(ATSM) complex in all the cancer cell lines tested. DNA interaction studies such as binding and cleavage showed that [Zn(GTSC)]3 was less harmful to DNA as well. All these features make [Zn(GTSC)]3 a good fluorescent imaging agent for live cells. Binuclear zinc bis(thiosemicarbazone) complexes were also synthesized and their cytotoxicity was evaluated in different cancer cells. One of the ligands, 1,3-bis{biacetyl-2′-"-N-pyrrolidinethiosemicarbazide)-3′-(4"-N-thiosemicarbazide)} propane (ProBATpyrH4), and its zinc complex were found to show excellent anticancer activity against human hepatocellular cancer (HepG2) cell line. However, the cellular uptake studies as followed by flow cytometry revealed that these compounds do not fluoresce inside the cells. However, the DNA interaction studies using ethidium bromide displacement assay revealed that these complexes have better binding ability to DNA than mononuclear zinc complexes and the viscometric titrations suggested the binding mode to DNA is through partial intercalation. Apparently, these complexes do not induce DNA cleavage as evident from the cleavage experiments performed on pBR322 DNA. It is likely that their anticancer activity is due to unique DNA binding properties. Imaging zinc is important in the field of metallomics as alteration of zinc concentration in cells is associated with, or attributed to various diseases. In this regard, bis(thiosemicarbazone) ligands are useful. Chapter 3, “Imaging intracellular zinc using glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) ligand” deals with imaging zinc in live cells using the bis(thiosemicarbazone) ligand, GTSCH2. Since the trimeric zinc complex is fluorescent, the corresponding ligand, GTSCH2, was utilized to visualize the zinc present within cells. The ligand GTSCH2 is found to be a selective fluorescence “turn-on” sensor for zinc. This sensor exhibited excellent sensitivity and selectivity towards zinc over other physiologically relevant cations. The binding affinity of GTSCH2 to zinc was estimated to be 0.59 nM in an aqueous MOPS (50 mM, NaCl; 100 mM; pH 7.3) buffer containing 30% DMSO, from competitive binding experiments carried out with ethylene glycol tetraacetic acid (EGTA). The sensor displayed maximal fluorescence response to zinc ion when present in the ratio of 1:1 and displayed stable fluoresence in the pH range 5.0 to 7.8, which suggests that the probe may be suitable for imaging zinc in both normal and cancer cells. The potential of GTSCH2 to image zinc inside the cell has been demonstrated in two human breast cancer cell lines using confocal fluorescence microscopy. Unlike mononuclear zinc complexes, the mononuclear copper bis(thiosemicarbazone) complexes are cytotoxic. Chapter 4, “Anticancer activity of copper bis(thiosemicarbazone) complexes” deals with the synthesis, characterization and anticancer activity of mononuclear copper bis(thiosemicarbazone) complexes. All of them were characterized by spectroscopic methods and in three cases by single crystal X-ray diffraction. The redox properties, studied by cyclic voltammetry, showed reversible one electron- reduction process that varied from –0.53 V to –0.18 V vsSCE. Anticancer activity for the synthesized complexes and their ligands were tested against many human cancer cell lines where the complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) are found to be most cytotoxic (GI50 <0.1 µM to 2.1 µM) in five cancer cell lines tested. Moreover, the cytotoxicity is similar to that of adriamycin, a known anticancer drug, in all cell lines. However, it is less potent than a copper bis(thiosemicarbazone) analog, copper biacetyl-bis(4-methyl-3-thiosemicarbazone) Cu(ATSM), a well studied anticancer agent in many cell lines. Cellular studies were carried out for the selected complexes Cu(GTSC) and Cu(GTSCHCl) along with Cu(ATSM) on HCT116 colon cancer cells. The order of lipophilicity and cellular uptake as studied by ICP-OES are correlated with their cytotoxicity. Based on the interaction of these complexes with DNA using the ethidium bromide displacement assay, DNA -melting, -viscosity and -cleavage studies, it is suggested that these complexes intercalate partially with DNA. DNA cleavage studies using pBR322 DNA revealed that only Cu(GTSCHCl) complex cleaves DNA. Mechanistic discrimination studies suggest that the complex cleaves DNA through the hydrolytic pathway. Since the topoisomerase IIα (Topo IIα), a nuclear enzyme resolving topological problems of DNA, is considered as one of the possible molecular targets for a number of anticancer drugs including some of the copper thiosemicarbazone complexes, Topo IIαinhibition studies were carried out in human Topo IIα. Interestingly, many copper bis(thiosemicarbazone) complexes are able to inhibit Topo IIα activity by acting as Topo IIα poison. Cu(GTSCHCl) complex was found to be the most active in this series of complexes (90 % inhibition at 100 µM) and it inhibits the enzyme in a dose dependant manner. Based on the results, it was concluded that the cell death may be mediated, at least in part, through DNA cleavage and Topo IIαinhibition. Severe side effects, poor distribution profiles and or organ specific toxicity make the conventional chemotherapy of limited value with metal based drugs. Therefore, developing cancer-specific drug delivery systems is an urgent need in cancer therapy. Among the many strategies available to target cancer, targeting the receptors that are overexpressed in the cancer cell membrane is a novel strategy being used in recent studies. Therefore the last part my work, “Copper bis(thiosemicarbazone) complexes linked to poly(ethylene glycol) and multiwalled carbon nanotubes for targeted delivery to cancer cells ” was designed to target cancer cells. Here, copper complexes (therapeutic molecule) were attached with PEG and MWCNT (carrier) along with folic acid or biotin (targeting molecule). First, CuATSM–A was functionalized with a disulfide linker and connected with folic acid via a poly(ethylene glycol) (PEG600) linker. This was synthesized to target KB (human nasopharyngeal carcinoma) cells, a cell line that overexpresses the folate receptor on the cell surface. In order to investigate the targeting efficacy, the corresponding fluorescent labeled analogs and non-targeted PEG conjugates were synthesized. Flow cytometry studies with fluorescent marker (fluorescein isothiocyanate) labeled PEG analogs showed the targeting efficacy on KB cells. The copper complex, CuATSM–A, attached with biotin–PEG2000 also was synthesized to target high-biotin-using HeLa (human cervical carcinoma) cells. Multiwalled carbon nanotubes (MWCNT) were also used as nanocarriers. Here, the MWCNT was decorated with PEG600 diamine and then functionalized with the copper complex (therapeutic molecule), folic acid (targeting molecule), and FITC (fluorescent molecule). The conjugation of all the molecules with MWCNT is characterized by various spectroscopic techniques.

<strong>Chapter 1</strong> Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. <strong>Chapter 2</strong> Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. <strong>Chapter 3</strong> Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. <strong>Chapter 4</strong> Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. <strong>Chapter 5</strong> Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB₂R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB₂R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.

A doença de Chagas, causada pelo protozoário flagelado Trypanosoma cruzi, é uma doença tropical que enseja morte/morbidade de milhões de pessoas na América Latina. Por processos migratórios, vem-se estendendo ao sul dos Estados Unidos, Canadá, Europa, Austrália e Japão. Essa doença tem sido considerada super-negligenciada pela indústria farmacêutica, já que os dois fármacos disponíveis para o seu tratamento foram introduzidos há mais de quarenta anos e apresentam baixa eficácia com vários efeitos colaterais severos. Mais recentemente, a Organização Mundial da Saúde considerou a doença de Chagas, dentre outras, como a doença da pobreza! Com esse cenário completamente desfavorável aos portadores da doença, é necessária a descoberta, desenvolvimento e introdução de novos fármacos para o tratamento eficiente e seguro da doença de Chagas. <br />Dentro desse contexto, este trabalho representa uma importante contribuição para o entendimento das razões moleculares da ação farmacológica de substâncias químicas bioativas de interesse à farmacoterapia da doença de Chagas. Ao nível molecular, a enzima pertencente à via de síntese de novo de nucleotídeos de pirimidinas, diidroorotato desidrogenase do Trypanosoma cruzi (TcDHODH), é um alvo promissor para a descoberta e desenvolvimento de candidatos a fármacos de interesse para o tratamento da doença de Chagas. <br />Os conceitos e ferramentas da química medicinal computacional, tais como os ensaios virtuais in silico, foram usados para a identificação de inibidores da TcDHODH. Vinte e seis substâncias inéditas como inibidores da TcDHODH foram adquiridos comercialmente e avaliados experimentalmente através da Calorimetria de Titulação Isotérmica (ITC) para a determinação do mecanismo de inibição e da constante cinética de afinidade (Kiapp). <br />Na etapa de docagem molecular, o objetivo era identificar moléculas que apresentassem uma boa afinidade pelo sítio ativo da enzima TcDHODH. A primeira série de ligantes selecionados dos métodos in silico, apresentou inibição enzimática na concentração de micromolar com eficiência média de ligante de 0,50 kcal mol-1 átomo-1. Devido à baixa massa molecular (aproximadamente 200 kDa) e a alta eficiência de ligante, essa série foi considerada como constituída de excelentes substâncias com elevado poder de reconhecimento biomolecular. Por isso, foram caracterizadas como substâncias passíveis de otimização no processo do-ligante-para-substância matriz. <br />As enzimas TcDHODH e DHODH de Leishmania major (LmDHODH) têm sítios ativos com elevado grau de similaridade. Portanto, usando a enzima LmDHODH como padrão de substituição da TcDHODH é possível fazer a descrição do modo de interação do co-complexo TcDHODH-inibidor. O modo de ação descrito através da resolução da estrutura cristalográfica de raios-X, além de validar ortogonalmente os resultados cinéticos obtidos por ITC - que identificou as substâncias como inibidores competitivos (por interação direta no sítio ativo da enzima TcDHODH), geraram hipóteses farmacofóricas para a busca de novas moléculas (chamadas de segunda geração), agora com padrão superior de reconhecimento molecular do sítio da TcDHODH. Para validar complementarmente a hipótese, foi demonstrado que os inibidores da TcDHODH inibem, similarmente, a LmDHODH. <br />Uma análise cuidadosa da estrutura tridimensional da enzima TcDHODH, demostrou a possibilidade de ocupação do sítio S2 que se estende além da região do sítio catalítico S1, permitindo assim o aumento da afinidade biomolecular com os inibidores. Além disso, o sítio S2 não é encontrado na estrutura da proteína de humanos (HsDHODH), podendo ser uma região passível de seletividade frente à enzima TcDHODH. <br />O emprego adequado dessa hipótese resultou na otimização dos ligantes identificados previamente para substâncias mais potentes que inibiram a enzima de forma competitiva em relação ao substrato diidroorotato (DHO) em valores Kiapp de 121 &plusmn; 14 nM e 190 &plusmn; 10 nM. <br />A técnica de ITC foi fundamental no processo de descoberta de inibidores enzimáticos, pois se mostrou extremamente susceptível à determinação da interação intermolecular enzima-inibidor, permitindo acompanhar a cinética da reação e obter os valores da constante de afinidade de maneira precisa e acurada. Com isso, a taxa de acerto obtida nesta tese foi de 46%, considerando-se apenas as substâncias com valores de Ki app < 100 &micro;M. Esse é um número favoravelmente apreciável, já que na literatura ele gira em torno de 1-10% quando o planejamento in silico é realizado, quando comparado às taxas de acerto dos métodos de ensaio em larga escala (HTS), entre 0-2 %, os resultados alcançados neste trabalho são ainda mais significativos. <br />Além disso, as substâncias químicas selecionadas através da integração de métodos in silico e biocalorimétricos apresentam elevado grau de complexidade no processo biomolecular de interação enzima-ligante, que permite classificá-las para as fases seguintes da gênese planejada de fármacos. / American trypanosomiasis or Chagas disease, caused by the haemoflagellate Trypanosoma cruzi, is a tropical disease that affects millions of people in Latin America. Epidemiology of Chagas disease in non-endemic countries is attained by immigration as the disease also affects people in the United States, Canada, Europe, Australia and Japan. However, the United States are not to be written off as an area of nonendemicity for Chagas disease like Europe or Asia because the southern states have enzootic T. cruzi transmission that involves triatomine species and hosts such as raccoons, opossums, and domestic dogs. Even though, this disease has been considered as a super-neglected from the big Pharma Industry viewpoint since the only available drugs for its treatment were introduced in the market more than forty years ago and worsen is that they have low efficacy and cause various severe side effects. <br />Although the current clinical scenario is of course discouraging and is far from being even a soothing treatment for those who suffer from the disease, it prompt ones to set efforts towards the need of discovering and developing new efficacious and safe drugs to treat Chagas disease. <br />Our research group covers the concept of enzymes acting as targets for the action of drugs. Once T. cruzi has many druggable targets, the dihydroorotate dehydrogenase enzyme (TcDHODH) that belongs to the de novo pyrimidine nucleotide synthetic pathway has been chosen for the search of new inhibitors that may be of use in the treatment of Chagas disease. To accomplish with this and considering that inhibitors are molecules that decrease enzyme activity leading to parasite death, we used the concepts and tools of modern computational medicinal chemistry such as in silico screening of small molecules that bind to the active site of the TcDHODH. <br />After a thoroughly program of virtually screening thousands of compounds, 26 were purchased from commercially available sources and experimentally assayed against the TcDHODH using Isothermal Titration Calorimetry (ITC) in order to determine the mechanism of inhibition and the kinetic affinity constant (Kiapp). <br />The first series of inhibitors selected from our in silico strategy were evaluated by ITC to yield compounds that inhibited the TcDHODH in the micromolar concentration range with an average of 0.50 kcal mol-1 atom-1 ligand efficiency (LE). Because the assayed compounds have low molecular weight (ca. 200 kDa) and high LE, which bring them to the specific bimolecular pattern recognition all of them were considered good inhibitors capable of being selected to enter the hit-to-lead optimization process. <br />The detailed description of the ligand-enzyme mode of binding (MOB) is thoroughly accomplished by solving the X ray crystal structure of the surrogate Leishmania major DHODH enzyme (LmDHODH), which has a high degree of similarity with the enzyme TcDHODH. The MOB credited to be in the active site of the TcDHODH orthogonally validated the ITC kinetic experimental data obtained for all ligands as competitive inhibitors that interact at the active site of the TcDHODH and helped to generate pharmacophoric hypotheses for the search of new second generation molecules acting against the enzyme TcDHODH.  Analyzing the 3D structure of the TcDHODH along with its surrogate LmDHODH, we envisaged the possibility of compounds to extend their side chain beyond the region of the catalytic site (called S1), and interacting in a region called S2, so to increase binding affinity. Moreover, the TcDHODH S2 site that is not found in the 3D protein structure of humans (HsDHODH) is likely to offer new insights for the search of inhibitors whose binding to this S2 site can pave the roads towards the needed structural basis for selective inhibition of TcDHODH. <br />The most potent compounds inhibited the enzyme competitively with respect to the substrate dihydroorotate (DHO) at Kiapp values of 121 &plusmn; 14 nM and 190 &plusmn; 10 nM, which constitutes high affinity TcDHODH inhibitors. The ITC technique was pivotal to this process of enzyme inhibitors discovery, because it proved to be extremely sensitive thus allowing to monitor the kinetics of the reaction and to obtain precise and accurate values of affinity constants. <br />The hit rate obtained in this work, considering only those compounds with Kiapp < 100 &micro;M, was 46%. This is a really high number, since literature values range from 1 to 10% when the planning new inhibitors via in silico methods when compared to the success rates obtained by the methods of testing on large scales (HTS), 0-2 %, the results achieved in this work are even more significant. Moreover, the compounds selected through the integration of in silico and calorimetric methods showed a high degree of complexity in the process of bimolecular enzyme-ligand recognition, which allows to pass them to the next phase of the drug design process.

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols. The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro were advanced. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and, potentially, in vivo applications. In order to further address the instability of AdoMet and analogs thereof, MAT-catalyzed reactions utilizing Met and ATP isosteres generated highly stable AdoMet isosteres that were capable of downstream utilization by MTs. Finally, the development, use, and results of a high-throughput screen identified mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.

Le développement d'une médecine personnalisée nécessite la mise au point de sondes permettant la détection des marqueurs d'une pathologie au stade le plus précoce possible. Ce mémoire décrit la synthèse et l'évaluation de sondes ciblées permettant de réaliser des images en IRM ou en scintigraphie. Ces sondes sont construites par fonctionnalisation d'un châssis moléculaire préparé par une réaction à trois composants. La propargylamine résultante possède plusieurs points d'ancrage pour introduire les ligands destinés au ciblage et deux groupes chélatants pour l'imagerie. Ce châssis possède aussi en position terminale un groupement réactif pour le fixer sur des petites molécules polyfonctionnelles, des dendrimères ou des protéines. Une technique de criblage à flux rapide, basée sur l'utilisation des puces à petites molécules, a été utilisée pour la détection des ligands. Les molécules déposées sur la puce proviennent soit des C-glycosides complexes préparés selon le concept de la synthèse orientée vers la diversité, soit de la chimiothèque du laboratoire. Ces puces ont permis l'identification de ligands de plusieurs protéines comme la protéine VPR de la capside du virus du sida. Un criblage secondaire par RMN a confirmé l'interaction protéine-petite molécule. Les sondes portant un flavonoïde pour cibler les cellules tumorales de l'endothélium ont été traitées par une solution de sel de gadolinium puis leurs propriétés ont été évaluées par IRM. Les premiers résultats indiquent que ces composés offrent un contraste de 2 à 4 fois supérieurs aux agents de contraste usuellement utilisés. Leur évaluation biologique sera réalisée prochainement.

El disseny molecular de sistemes d’interès per a la química mèdica i per al disseny de fàrmacs sempre s’ha trobat molt lligat a la disponibilitat sintètica dels resultats. Des del moment que la química combinatòria s’incorpora dins de l’esquema sintètic, canvia el paper que ha de jugar la química computacional: la diversitat d’estructures possibles a sintetitzar fa necessària la introducció de mètodes, com el cribratge virtual, que permetin avaluar la viabilitat de grans quimioteques virtuals amb un temps raonable. Els mètodes quimioinformàtics responen a la necessitat anterior, posant a l’abast de l’usuari mètodes eficaços per a la predicció teòrica d’activitats biològiques o propietats d’interès. Dins d’aquests destaquen els mètodes basats en la relació quantitativa d’estructura-activitat (QSAR). Aquests han demostrat ser eficaços per l’establiment de models de predicció en l’àmbit farmacològic i biomèdic. S’ha avaluat la utilització de mètodes QSAR no lineals en la teràpia fotodinàmica del càncer, donat que és una de les línies de recerca d’interès del Grup d’Enginyeria Molecular (GEM) de l’IQS. El disseny de fotosensibilitzadors es pot realitzar a partir de la predicció de propietats fisicoquímiques (com l’espectre d’absorció i la hidrofobicitat del sistema molecular), i de l’estudi de la seva localització subcel•lular preferent, la qual ha demostrat recentment jugar un paper molt important en l’eficàcia del procés global. Per altra banda, les xarxes neuronals artificials són actualment un dels mètodes més ben valorats per a l’establiment de models QSAR no lineals. Donat l’interès de disposar d’un programari capaç d’aplicar aquests mètodes i que, a més, sigui prou versàtil i adaptable com per poder-se aplicar a diferents problemes, s’ha desenvolupat el programari ArIS. Aquest inclou els principals mètodes de xarxes neuronals artificials, per realitzar tasques de classificació i predicció quantitativa, necessaris per a l’estudi de problemes d’interès, com és la predicció de l’activitat anti-VIH d’anàlegs de l’AZT, l’optimització de formulacions químiques o el reconeixement estructural de grans sistemes moleculars / El diseño molecular de sistemas de interés para la química médica y para el diseño de fármacos siempre ha estado condicionado por la disponibilidad sintética de los resultados. Desde el momento en que la química combinatoria se incorpora en el esquema sintético, cambia el papel de la química computacional: la diversidad de estructuras que pueden sintetizarse hace necesaria la introducción de métodos, como el cribado virtual, que permitan evaluar la viabilidad de grandes quimiotecas virtuales en un tiempo razonable. Los métodos quimioinformáticos responden a la necesidad anterior, ofreciendo al usuario métodos eficaces para la predicción teórica de actividades biológicas o propiedades de interés. Entre ellos destacan los métodos basados en la relación cuantitativa de estructura-actividad (QSAR), que han demostrado ser eficaces para establecer modelos de predicción en el ámbito farmacológico y biomédico. Se ha evaluado la utilización de métodos QSAR no lineales en terapia fotodinámica del cáncer, dado que es una de las líneas de investigación de interés del Grup d’Enginyeria Molecular (GEM) del IQS. El diseño de fotosensibilizadores se puede realizar a partir de la predicción de propiedades fisicoquímicas (como su espectro de absorción o su hidrofobicidad) y del estudio de su localización subcelular preferente, la cual ha demostrado recientemente jugar un papel muy importante en la eficacia del proceso global. Por otro lado, las redes neuronales artificiales son actualmente uno de los métodos mejor valorados para establecer modelos QSAR no lineales. Es por ello que resulta muy interesante disponer de un programa capaz de aplicar estos métodos y que, además, sea lo suficientemente versátil y adaptable como para poder aplicarse a distintos problemas, según las necesidades del usuario. Por este motivo se ha desarrollado el programa ArIS, el cual incluye los principales métodos de redes neuronales artificiales para realizar tareas de clasificación y predicción cuantitativa, necesarios para el estudio de problemas de interés como la predicción de la actividad anti-VIH de análogos del AZT, la optimización de formulaciones químicas o el reconocimiento estructural de grandes sistemas moleculares. / Molecular modelling of interesting systems for medicinal chemistry and drug design highly depends on availability of synthetic results. Since combinatorial chemistry was incorporated into the synthetic scheme, the role of computational chemistry has changed: the structural diversity of candidates to be synthesized requires the introduction of computational methods which are able to screen large virtual libraries. Answering to this requirement, chemoinformatics offers many kinds of different methods for predicting biological activities and molecular properties. One of the most relevant techniques among them is Quantitative Structure-Activity Relationships (QSAR), which can be used to establish prediction models for both, pharmacological and biomedical sectors. The use of non- linear QSAR methods has been evaluated in photodynamic therapy of cancer, one of the research areas of the Grup d’Enginyeria Molecular (GEM) at IQS. Molecular design of photosensitizers can be performed by computational studies of their physicochemical properties (absorption spectra or hydrophobicity, for example) and subcellular localization, which becomes a key factor in the efficacy of the overall process. Furthermore, artificial neural networks are nowadays rated as one of the very best methods for establishing non-linear QSAR models. Developing software that includes all these methods would be certainly interesting. Implemented algorithms should be versatile and easily adaptable for their use in any problems. We have developed ArIS software, which includes the most important methods of artificial neural networks for classification and quantitative prediction. ArIS has been used to predict anti-HIV activity of AZT-analogues, for optimization of chemical formulations and for structural recognition in large molecular systems, among others.

The dissertation describes a novel method for plant drug discovery based on mutation and selection of plant cells. Despite the industry focus on chemical synthesis, plants remain a source of potent and complex bioactive metabolites. Many of these have evolved as defensive compounds targeted on key proteins in the CNS of herbivorous insects, for example the insect dopamine transporter (DAT). Because of homology with the human DAT protein some of these metabolites have high abuse potential, but others may be valuable in treating drug dependence. This dissertation redirects the evolution of a native Lobelia species toward metabolites with greater activity at this therapeutic target, i.e. the human DAT. This was achieved by expressing the human DAT protein in transgenic plant cells and selecting gain-of-function mutants for survival on medium containing a neurotoxin that is accumulated by the human DAT. This created a sub-population of mutants with increased DAT inhibitory activity. Some of the active metabolites in these mutants are novel (i.e. not detectable in wild-type cells). Others are cytoprotective, and also protect DAergic neurons against the neurotoxin. This provides proof-of-concept for a novel plant drug discovery platform, which is applicable to many different therapeutic target proteins and plant species.

A doença de Chagas, causada pelo protozoário flagelado Trypanosoma cruzi, é uma doença tropical que enseja morte/morbidade de milhões de pessoas na América Latina. Por processos migratórios, vem-se estendendo ao sul dos Estados Unidos, Canadá, Europa, Austrália e Japão. Essa doença tem sido considerada super-negligenciada pela indústria farmacêutica, já que os dois fármacos disponíveis para o seu tratamento foram introduzidos há mais de quarenta anos e apresentam baixa eficácia com vários efeitos colaterais severos. Mais recentemente, a Organização Mundial da Saúde considerou a doença de Chagas, dentre outras, como a doença da pobreza! Com esse cenário completamente desfavorável aos portadores da doença, é necessária a descoberta, desenvolvimento e introdução de novos fármacos para o tratamento eficiente e seguro da doença de Chagas. <br />Dentro desse contexto, este trabalho representa uma importante contribuição para o entendimento das razões moleculares da ação farmacológica de substâncias químicas bioativas de interesse à farmacoterapia da doença de Chagas. Ao nível molecular, a enzima pertencente à via de síntese de novo de nucleotídeos de pirimidinas, diidroorotato desidrogenase do Trypanosoma cruzi (TcDHODH), é um alvo promissor para a descoberta e desenvolvimento de candidatos a fármacos de interesse para o tratamento da doença de Chagas. <br />Os conceitos e ferramentas da química medicinal computacional, tais como os ensaios virtuais in silico, foram usados para a identificação de inibidores da TcDHODH. Vinte e seis substâncias inéditas como inibidores da TcDHODH foram adquiridos comercialmente e avaliados experimentalmente através da Calorimetria de Titulação Isotérmica (ITC) para a determinação do mecanismo de inibição e da constante cinética de afinidade (Kiapp). <br />Na etapa de docagem molecular, o objetivo era identificar moléculas que apresentassem uma boa afinidade pelo sítio ativo da enzima TcDHODH. A primeira série de ligantes selecionados dos métodos in silico, apresentou inibição enzimática na concentração de micromolar com eficiência média de ligante de 0,50 kcal mol-1 átomo-1. Devido à baixa massa molecular (aproximadamente 200 kDa) e a alta eficiência de ligante, essa série foi considerada como constituída de excelentes substâncias com elevado poder de reconhecimento biomolecular. Por isso, foram caracterizadas como substâncias passíveis de otimização no processo do-ligante-para-substância matriz. <br />As enzimas TcDHODH e DHODH de Leishmania major (LmDHODH) têm sítios ativos com elevado grau de similaridade. Portanto, usando a enzima LmDHODH como padrão de substituição da TcDHODH é possível fazer a descrição do modo de interação do co-complexo TcDHODH-inibidor. O modo de ação descrito através da resolução da estrutura cristalográfica de raios-X, além de validar ortogonalmente os resultados cinéticos obtidos por ITC - que identificou as substâncias como inibidores competitivos (por interação direta no sítio ativo da enzima TcDHODH), geraram hipóteses farmacofóricas para a busca de novas moléculas (chamadas de segunda geração), agora com padrão superior de reconhecimento molecular do sítio da TcDHODH. Para validar complementarmente a hipótese, foi demonstrado que os inibidores da TcDHODH inibem, similarmente, a LmDHODH. <br />Uma análise cuidadosa da estrutura tridimensional da enzima TcDHODH, demostrou a possibilidade de ocupação do sítio S2 que se estende além da região do sítio catalítico S1, permitindo assim o aumento da afinidade biomolecular com os inibidores. Além disso, o sítio S2 não é encontrado na estrutura da proteína de humanos (HsDHODH), podendo ser uma região passível de seletividade frente à enzima TcDHODH. <br />O emprego adequado dessa hipótese resultou na otimização dos ligantes identificados previamente para substâncias mais potentes que inibiram a enzima de forma competitiva em relação ao substrato diidroorotato (DHO) em valores Kiapp de 121 &plusmn; 14 nM e 190 &plusmn; 10 nM. <br />A técnica de ITC foi fundamental no processo de descoberta de inibidores enzimáticos, pois se mostrou extremamente susceptível à determinação da interação intermolecular enzima-inibidor, permitindo acompanhar a cinética da reação e obter os valores da constante de afinidade de maneira precisa e acurada. Com isso, a taxa de acerto obtida nesta tese foi de 46%, considerando-se apenas as substâncias com valores de Ki app < 100 &micro;M. Esse é um número favoravelmente apreciável, já que na literatura ele gira em torno de 1-10% quando o planejamento in silico é realizado, quando comparado às taxas de acerto dos métodos de ensaio em larga escala (HTS), entre 0-2 %, os resultados alcançados neste trabalho são ainda mais significativos. <br />Além disso, as substâncias químicas selecionadas através da integração de métodos in silico e biocalorimétricos apresentam elevado grau de complexidade no processo biomolecular de interação enzima-ligante, que permite classificá-las para as fases seguintes da gênese planejada de fármacos. / American trypanosomiasis or Chagas disease, caused by the haemoflagellate Trypanosoma cruzi, is a tropical disease that affects millions of people in Latin America. Epidemiology of Chagas disease in non-endemic countries is attained by immigration as the disease also affects people in the United States, Canada, Europe, Australia and Japan. However, the United States are not to be written off as an area of nonendemicity for Chagas disease like Europe or Asia because the southern states have enzootic T. cruzi transmission that involves triatomine species and hosts such as raccoons, opossums, and domestic dogs. Even though, this disease has been considered as a super-neglected from the big Pharma Industry viewpoint since the only available drugs for its treatment were introduced in the market more than forty years ago and worsen is that they have low efficacy and cause various severe side effects. <br />Although the current clinical scenario is of course discouraging and is far from being even a soothing treatment for those who suffer from the disease, it prompt ones to set efforts towards the need of discovering and developing new efficacious and safe drugs to treat Chagas disease. <br />Our research group covers the concept of enzymes acting as targets for the action of drugs. Once T. cruzi has many druggable targets, the dihydroorotate dehydrogenase enzyme (TcDHODH) that belongs to the de novo pyrimidine nucleotide synthetic pathway has been chosen for the search of new inhibitors that may be of use in the treatment of Chagas disease. To accomplish with this and considering that inhibitors are molecules that decrease enzyme activity leading to parasite death, we used the concepts and tools of modern computational medicinal chemistry such as in silico screening of small molecules that bind to the active site of the TcDHODH. <br />After a thoroughly program of virtually screening thousands of compounds, 26 were purchased from commercially available sources and experimentally assayed against the TcDHODH using Isothermal Titration Calorimetry (ITC) in order to determine the mechanism of inhibition and the kinetic affinity constant (Kiapp). <br />The first series of inhibitors selected from our in silico strategy were evaluated by ITC to yield compounds that inhibited the TcDHODH in the micromolar concentration range with an average of 0.50 kcal mol-1 atom-1 ligand efficiency (LE). Because the assayed compounds have low molecular weight (ca. 200 kDa) and high LE, which bring them to the specific bimolecular pattern recognition all of them were considered good inhibitors capable of being selected to enter the hit-to-lead optimization process. <br />The detailed description of the ligand-enzyme mode of binding (MOB) is thoroughly accomplished by solving the X ray crystal structure of the surrogate Leishmania major DHODH enzyme (LmDHODH), which has a high degree of similarity with the enzyme TcDHODH. The MOB credited to be in the active site of the TcDHODH orthogonally validated the ITC kinetic experimental data obtained for all ligands as competitive inhibitors that interact at the active site of the TcDHODH and helped to generate pharmacophoric hypotheses for the search of new second generation molecules acting against the enzyme TcDHODH.  Analyzing the 3D structure of the TcDHODH along with its surrogate LmDHODH, we envisaged the possibility of compounds to extend their side chain beyond the region of the catalytic site (called S1), and interacting in a region called S2, so to increase binding affinity. Moreover, the TcDHODH S2 site that is not found in the 3D protein structure of humans (HsDHODH) is likely to offer new insights for the search of inhibitors whose binding to this S2 site can pave the roads towards the needed structural basis for selective inhibition of TcDHODH. <br />The most potent compounds inhibited the enzyme competitively with respect to the substrate dihydroorotate (DHO) at Kiapp values of 121 &plusmn; 14 nM and 190 &plusmn; 10 nM, which constitutes high affinity TcDHODH inhibitors. The ITC technique was pivotal to this process of enzyme inhibitors discovery, because it proved to be extremely sensitive thus allowing to monitor the kinetics of the reaction and to obtain precise and accurate values of affinity constants. <br />The hit rate obtained in this work, considering only those compounds with Kiapp < 100 &micro;M, was 46%. This is a really high number, since literature values range from 1 to 10% when the planning new inhibitors via in silico methods when compared to the success rates obtained by the methods of testing on large scales (HTS), 0-2 %, the results achieved in this work are even more significant. Moreover, the compounds selected through the integration of in silico and calorimetric methods showed a high degree of complexity in the process of bimolecular enzyme-ligand recognition, which allows to pass them to the next phase of the drug design process.

The present thesis deals with different aspects of the chemistry of oxovanadium(IV) complexes, their interaction with double stranded DNA, photo-induced DNA cleavage, photo-enhanced cytotoxicity in visible light and red light and localisation and cellular uptake to understand the mechanism of cell death. Chapter I presents a general introduction on potential of transition metal complexes as photochemotherapeutic agents. A brief introduction about Photodynamic Therapy (PDT) as a new alternative to chemotherapy for treating cancer has been made. Various modes of interaction of small molecules with duplex DNA are described. Recent reports on metal-based photocytotoxicity, photo-induced DNA cleavage activity and cellular localization are presented in detail. Objective of the present investigation is also dealt in this Chapter. Chapter II of the thesis deals with the synthesis, characterization, DNA binding and photo-induced DNA cleavage activity of ternary oxovanadium(IV) complexes of ONO-donor 2-(2-hydroxybenzylideneamino)phenol (salamp) and phenanthroline bases to explore the photo-induced DNA cleavage activity in UV-A light of 365 nm and photocytotoxicity in visible light. Chapter III deals with the photo-induced DNA cleavage and photocytotoxicity of ternary oxovanadium(IV) complexes containing ONN-donor N-2-pyridylmethylidine-2-hydroxyphenylamine (Hpyamp) Schiff bases and phenanthroline bases. The objective of this work is to investigate the photo-induced DNA cleavage activity in near-IR light. Photocytotoxicity and cell cycle arrest have been studied in HeLa cancer cells. Chapter IV deals serendipitous discovery of planar triazinuim cationic species by vanadyl-assisted novel ring cyclization reaction. The compounds are synthesised, characterized and their DNA binding and anaerobic photoinduced DNA cleavage activity are presented. The importance of the thiazole moiety in the triazinuim species in cellular uptake has been investigated. Photocytotoxicity, localization and cell death mechanism have been studied in HeLa and MCF-7 cells. Chapter V describes the synthesis, characterization, DNA binding, photo-induced DNA cleavage activity and photocytotoxicity of oxovanadium(IV) complexes containing 2-(1H-benzimidazol-2-yl)-N-(pyridin-2-ylmethylene)ethaneamine (Hpy-aebmz) and curcumin as photosensitizer. The effect of conjugating naphthalimide on Hpy-aebmz on photoinduced DNA cleavage and photocytotoxicity has been studied. Cellular uptake, localization and mechanism of cell death induced by complexes have been investigated. Chapter VI presents ternary oxovanadium(IV) complexes having, 2-((1H-benzimidazol-2-yl)methylimino-methyl)phenol (Hsal-ambmz) and phenanthroline bases. The complexes were synthesized, characterized and their DNA binding property studied. Photo-induced DNA cleavage activity and photocytotoxicity in red light has been discussed. Anthracene has been conjugated to a tridentate ligand to investigate cellular uptake, localization and cell death mechanism. Mitochondria targeting property of the complexes having dipeptide has been studied and compared with clinically used drug Photofrin®. The references have been compiled at the end of each chapter and indicated as superscript numbers in the text. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the complexes, characterized structurally by single crystal X-ray crystallography, are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight or mistake is regretted.