ARTIFICIAL NEURAL NETWORKS: FUNCTIONINGANDAPPLICATIONS IN PHARMACEUTICAL INDUSTRY

Artificial Neural Network (ANN) technology is a group of computer designed algorithms for simulating neurological processing to process information and produce outcomes like the thinking process of humans in learning, decision making and solving problems. The uniqueness of ANN is its ability to deliver desirable results even with the help of incomplete or historical data results without a need for structured experimental design by modeling and pattern recognition. It imbibes data through repetition with suitable learning models, similarly to humans, without actual programming. It leverages its ability by processing elements connected with the user given inputs which transfers as a function and provides as output. Moreover, the present output by ANN is a combinational effect of data collected from previous inputs and the current responsiveness of the system. Technically, ANN is associated with highly monitored network along with a back propagation learning standard. Due to its exceptional predictability, the current uses of ANN can be applied to many more disciplines in the area of science which requires multivariate data analysis. In the pharmaceutical process, this flexible tool is used to simulate various non-linear relationships. It also finds its application in the enhancement of pre-formulation parameters for predicting physicochemical properties of drug substances. It also finds its applications in pharmaceutical research, medicinal chemistry, QSAR study, pharmaceutical instrumental engineering. Its multi-objective concurrent optimization is adopted in the drug discovery process, protein structure, rational data analysis also

Towards intelligent drug design system: Application of artificial dipeptide receptor library in QSAR-oriented studies

The pharmacophore properties of a new series of potential purinoreceptor (P2X) inhibitors determined using a coupled neural network and the partial least squares method with iterative variable elimination (IVE-PLS) are presented in a ligand-based comparative study of the molecular surface by comparative molecular surface analysis (CoMSA). Moreover, we focused on the interpretation of noticeable variations in the potential selectiveness of interactions of individual inhibitor-receptors due to their physicochemical properties; therefore, the library of artificial dipeptide receptors (ADP) was designed and examined. The resulting library response to individual inhibitors was arranged in the array, preprocessed and transformed by the principal component analysis (PCA) and PLS procedures. A dominant absolute contribution to PC1 of the Glu attached to heptanoic gating acid and Phe bonded to the linker m-phenylenediamine/triazine scaffold was revealed by the PCA. The IVE-PLS procedure indicated the receptor systems with predominant Pro bonded to the linker and Glu, Gln, Cys and Val directly attached to the gating acid. The proposed comprehensive ligand-based and simplified structure-based methodology allows the in-depth study of the performance of peptide receptors against the tested set of compounds.NC

Proline-based carbamates as cholinesterase inhibitors

Series of twenty-five benzyl (2S)-2-(arylcarbamoyl)pyrrolidine-1-carboxylates was prepared and completely characterized. All the compounds were tested for their in vitro ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and the selectivity of compounds to individual cholinesterases was determined. Screening of the cytotoxicity of all the compounds was performed using a human monocytic leukaemia THP-1 cell line, and the compounds demonstrated insignificant toxicity. All the compounds showed rather moderate inhibitory effect against AChE; benzyl (2S)-2-[(2-chlorophenyl)carbamoyl]pyrrolidine-1-carboxylate (IC50 = 46.35 M) was the most potent agent. On the other hand, benzyl (2S)-2-[(4-bromophenyl)-] and benzyl (2S)-2-[(2-bromophenyl)carbamoyl]pyrrolidine-1-carboxylates expressed anti-BChE activity (IC50 = 28.21 and 27.38 M, respectively) comparable with that of rivastigmine. The ortho-brominated compound as well as benzyl (2S)-2-[(2-hydroxyphenyl)carbamoyl]pyrrolidine-1-carboxylate demonstrated greater selectivity to BChE. The in silico characterization of the structure–inhibitory potency for the set of proline-based carbamates considering electronic, steric and lipophilic properties was provided using comparative molecular surface analysis (CoMSA) and principal component analysis (PCA). Moreover, the systematic space inspection with splitting data into the training/test subset was performed to monitor the statistical estimators performance in the effort to map the probability-guided pharmacophore pattern. The comprehensive screening of the AChE/BChE profile revealed potentially relevant structural and physicochemical features that might be essential for mapping of the carbamates inhibition efficiency indicating qualitative variations exerted on the reaction site by the substituent in the 30-/40-position of the phenyl ring. In addition, the investigation was completed by a molecular docking study of recombinant human AChE

The anti-proliferative activity of drimia altissima and a novel isolated flavonoid glycoside against hela cervical cancer cells

Cancer is one of the leading causes of mortality worldwide. About 44% of all cancer morbidity and 53% of all cancer mortality occur in countries with a low to medium Human Development Index (HDI). Thus, cancer is rapidly emerging as a serious threat to public health in Africa and most especially, sub-Saharan Africa. The International Agency for Research on Cancer (IARC) projects that there will be 1.28 million new cancer cases and 970 000 cancer deaths in Africa by the year 2030 owing to the increase in economic development associated lifestyles. The dominant types of cancer in Africa are those related to infectious diseases such as Kaposi’s sarcoma and cervical, hepatic and urinary bladder carcinomas. The main challenge to cancer treatment in Africa is the unavailability of efficacious anticancer drugs. This is because most developing countries can only afford to procure the most basic anticancer drugs, which are also frequently unavailable due to intermittent supplies. This results in patients progressing to more advanced cancer states. One way of combating this African problem is to focus on research that aims at discovering efficacious and cost effective cancer therapies from available natural resources within the African continent. This study investigated the potential anti-proliferative activity (against HeLa cervical cancer cells) of four plants (Adansonia digitata, Ceiba pentandra, Maytenus senegalensis and Drimia altissima) commonly used in the African traditional treatment of malignancies. After in vitro bio-assay screening using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, M. senegalensis root extract (MS-R) and D. altissima bulb extract (DA-B) showed anti-proliferative activity against HeLa cervical cancer cells with IC50 values of 25 μg/mL and 1.1 μg/mL respectively. By possessing the strongest anti-proliferative activity among the tested extracts, D. altissima was selected for further studies. Liquid-liquid partitioning of the Drimia altissima bulb extract with n-hexane, ethyl acetate, and n-butanol, yielded partitions 79a – d, with the n-butanol fraction, 79d, exhibiting the strongest cytotoxic activity (IC50 = 0.497 μg/mL). Through High Content Analysis (HCA) screening, fraction 79d was found to induce marked early mitotic cell cycle arrest. Fractionation of 79d using Diaion® HP-20 open column chromatography and a stepwise gradient of reducing polarity (water-methanol-ethanol-ethyl acetate) yielded cytotoxic fractions 82b, 82c, 82d and 82e, all with significant anti-proliferative activities at the tested concentrations of 0.1, 1.0 and 10 μg/mL. Bio-assay guided fractionation of 82c (the most effective fraction at the lowest tested concentration of 0.1 μg/mL) using Sephadex® LH-20 open column chromatography and 50% MeOH led to the isolation of compound 3.17. After structural elucidation using 1D and 2D Nuclear Magnetic Resonance spectroscopy (NMR), High resolution Mass spectrometry (HRMS), Fourier-Transform Infrared spectroscopy (FT-IR), ultraviolet spectroscopy (UV) and Circular Dichroism (CD), compound 3.17 was identified as a novel C-glucosylflavonoid-O-glucoside, 6-C-[-apio-α-D-furanosyl-(1→6)-β-glucopyranosyl]-4′, 5, 7-trihydroxyflavone (Altissimin, 3.17). Compound 3.17 exhibited a dose dependant anti-proliferative activity with an IC50 of 2.44 μM. The mechanism of action for compound 3.17 was investigated through cell cycle arrest, phosphatidylserine translocation (PS), caspase activation and mitochondrial membrane depolarization. The mechanism of cell death elicited by compound 3.17 in HeLa cells was found to involve the induction of M phase cell cycle arrest with consequent activation of apoptotic cell death which was evident from annexin V staining, mitochondrial membrane potential (ΔΨm) collapse and the activation of caspases -8 and -3. In silico computational techniques were employed to virtually determine potential biological targets of compound 3.17. Target fishing using the Similarity Ensemble Approach (SEA) target prediction gave human aldose reductase (hAR, AKR1B1) the highest ranking with a p value of 2.85 x 10-24, a max Tc of 0.35 and a Z-score of 41.8217. Using AutoDock4 and the AutoDock tools suite (ADT), molecular docking of compound 3.17 in the hAR binding pocket was successfully achieved with a lower ΔG free energy binding (-9.4 kcal/mol) than that of positive control ligand 393 (-8.7 kcal/mol). In conclusion, this study identified the genus Drimia and particularly D. altissima as a potential source for novel cytotoxic compounds. The discovery of altissimin (3.17), the first flavonoid glycoside to be isolate from D. altissima, enquires into the possible existence of similar compounds within the species. In addition to the observed in vitro cytotoxic activity against HeLa cells, the potential of altissimin (3.17) as a hAR enzyme inhibitor opens up the possibility of its use as an adjunct to increase cancer cell sensitivity to chemotherapy. Thus, altissimin (3.17) shows promise as a potential anticancer agent

Hsp90 as a molecular target

Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, has been proposed to play a vital role in tumorigenesis. Hsp90 has two isoforms, of which Hsp90α is the major isoform of the Hsp90 complex and has an inducible expression profile. The molecular chaperone Hsp90α has been recognized in different cancers and it is implicated to play a role in cell cycle progression, apoptosis, regulates invasion, angiogenesis and metastasis. It is being recognized as a promising target in cancer treatment. Previous studies in our laboratory have demonstrated hsp90α expression in both primary glioma tissue and cell lines, but not in normal healthy brain tissues and cell lines. Enhanced chemosensitivity was observed upon specific inhibition of hsp90α expression by siRNA, suggesting that inhibiting hsp90α expression could possibly be a favourable therapeutic approach compared to conventional chemotherapies. In this novel study, Hsp90 was inhibited by either treatment with 17AAG or shRNA oligonucleotide targeting hsp90α (shhsp90α) in the U87-MG glioma cell line. The inhibition profile of Hsp90α was observed at the protein levels in control and treated cells by FACS analysis (quantitative) using a flow cytometer and Hsp90α ELISA kit. The results demonstrated a significant reduction of Hsp90α protein levels post treatment with 17AAG and shhsp90α. The activity of Hsp90α was assayed by quantifying the levels of Akt/PKB in the samples. Significant reductions (>50 %) of Akt/PKB levels were observed post hsp90α inhibition. Cell cycle analysis carried out reported S and G2 phase arrest, post Hsp90 inhibition by either 17AAG or shhsp90α. Interestingly, it was reported that 17AAG shows a better silencing profile compared to shhsp90α. To analyse the downstream effects of Hsp90 inhibition and to determine the client proteins affected, proteomic analysis was performed. Proteomic analysis identified several proteins which were either upregulated/downregulated post Hsp90 inhibition. IPA analysis further identified “cancer” as the top network significantly transformed post Hsp90 inhibition. Upregulated proteins include Hsp70 family members, Hsp27 and gp96, thereby suggesting the role of Hsp90 co-chaperones in compensating for Hsp90 function post Hsp90 inhibition. Moreover, members of the glycolysis/glucogenesis pathway were also upregulated, demonstrating increased dependency on glycolysis for energy supply by the treated glioma cells. Considering Hsp70 and its role in anti-apoptosis, it was postulated that a combination therapy involving a multi-target approach could be carried out. Subsequently, inhibition of both Hsp90 and Hsp70 in U87-MG glioma cell line was carried out resulting in 60 % cell death along with S and G2 phase arrest. Thus, in the effective treatment of glioma, the inhibition of multiple targets needs to be taken into consideration. Conclusion: It can be thus concluded that, combination therapy involving silencing of Hsp90 and Hsp70 could be of possible significance in glioma therapy

INTEGRATING CHEMICAL, BIOLOGICAL AND PHYLOGENETIC SPACES OF AFRICAN NATURAL PRODUCTS TO UNDERSTAND THEIR THERAPEUTIC ACTIVITY

INTEGRATING CHEMICAL, BIOLOGICAL AND PHYLOGENETIC SPACES OF AFRICAN NATURAL PRODUCTS TO UNDERSTAND THEIR THERAPEUTIC ACTIVITY Fatima Magdi Hamza Baldo This research aims to utilise ligand-based target prediction to (i) understand the mechanism of action of African natural products (ANPs), (ii) help identify patterns of phylogenetic use in African traditional medicine and (iii) elucidate the mechanism of action of phenotypically active small molecules and natural products with anti-trypanosomal activity. In Chapter 2 the objective was to utilise ligand-based target prediction to understand the mechanism of action of natural products (NPs) from African medicinal plants used against cancer. The Random Forest classifier used in this work compares the similarity of the input compounds from the natural product dataset with compound-target combinations in the training set. The more similar they are in structure, the more likely they are to modulate the same target. Natural products from plants used against cancer in Africa were predicted to modulate targets and pathways directly associated with the disease, thus understanding their mechanism of action e.g. “flap endonuclease 1” and “Mcl-1”. The “Keap1-Nrf2 Pathway” and “apoptosis modulation by HSP70”, two pathways previously linked to cancer (which are not currently targeted by marketed drugs, but have been of increasing interest in recent years) were predicted to be modulated by ANPs. In Chapter 3, we aimed to identify phylogenetic patterns in medicinal plant use and the role this plays in predicting medicinal activity. We combined chemical, predicted target and phylogenetic information of the natural products to identify patterns of use for plant families containing plant species used against cancer in African, Malay and Indian (Ayurveda) traditional medicine. Plant families that are close phylogenetically were found to produce similar natural products that act on similar targets regardless of their origin. Additionally, phylogenetic patterns were identified for African traditional plant families with medicinal species used against cancer, malaria and human African trypanosomiasis (HAT). We identified plant families that have more medicinal species than would statistically be expected by chance and rationalised this by linking their activity to their unique phyto-chemistry e.g. the napthyl-isoquinoline alkaloids, uniquely produced by Acistrocladaceae and Dioncophyllaceae, are responsible for anti-malarial and anti-trypanosome activity. In Chapter 4, information from target prediction and experimentally validated targets was combined with orthologue data to predict targets of phenotypically active small molecules and natural products screened against Trypanosoma brucei. The predicted targets were prioritised based on their essentiality for the survival of the T. brucei parasite. We predicted orthologues of targets that are essential for the survival of the trypanosome e.g. glycogen synthase kinase 3 (GSK3) and rhodesain. We also identified the biological processes predicted to be perturbed by the compounds e.g. “glycolysis”, “cell cycle”, “regulation of symbiosis, encompassing mutualism through parasitism” and “modulation of development of symbiont involved in interaction with host”. In conclusion, in silico target prediction can be used to predict protein targets of natural products to understand their molecular mechanism of action. Phylogenetic information and phytochemical information of medicinal plants can be integrated to identify plant families with more medicinal species than would be expected by chance

Construcción QSAR de redes complejas de compuestos de interés en Química Farmacéutica, Microbiología y Parasitología

El diseño para la búsqueda y desarrollo de fármacos eficaces para el tratamiento de estas enfermedades, que supriman la eliminación o la degeneración celular respectivamente, es una de las líneas de investigación más importantes dentro de la química farmacéutica. En esto entra el diseño de fármacos; el diseño de fármacos está dedicado al desarrollo de modelos matemáticos para predecir propiedades de interés para una gran variedad de sistemas químicos incluyendo moléculas de bajo peso molecular, polímeros, biopolímeros, sistemas heterogéneos, formulaciones farmacéuticas, conglomerados de moléculas e iones, materiales, nano-estructuras y otros. Este tipo de predicciones no pretenden sustituir las técnicas experimentales sino complementar las mismas ayudando a obtener nuevas moléculas activas con mayor probabilidad de éxito, con la ventaja que ello supone en términos de ahorro de tiempo, recursos materiales, y muy importante: el refinamiento y reducción en el uso de animales de laboratorio. Esta metodología se basa en el uso de cálculos por ordenador y en las nuevas tecnologías de la informática. Las cuales pueden ser usadas: Para moléculas pequeñas: a) Estudios de relación cuantitativa estructura molecular-actividad farmacológica (QSAR) y de estructura molecular propiedades toxicológicas y eco-toxicológicas incluyendo mutagenicidad e carcinogénesis (QSTR). b) Predicción de propiedades químicas y fisicoquímicas de moléculas. Estudios de relación estructura molecular y propiedades de absorción, distribución, metabolismo y eliminación (ADME). c) Predicción de mecanismos de acción biológica de moléculas y evaluación in sílico de alta eficacia para grandes bases de datos (virtual HTS). Para macromoléculas: a) Estudios de interacción fármaco-receptor (neuronas). b) Bioinformática aplicada a estudios de relación secuencia-función y propiedades estructurales de ácidos nucleicos y proteínas. c) Búsqueda de nuevas dianas terapéuticas y “sitio activo” a partir de datos de Genómica, Proteómica. d) Búsqueda de biomarcadores para diagnóstico de enfermedades o como indicadores de contaminaciones. e) Predicción de propiedades fisicoquímicas de polímeros sintéticos, biopolímeros, materiales y nano-estructuras. f) Predicción, diseño, y optimización de enzimas mutadas para procesos biotecnológicos

Bases moleculares da galactosémia clássica : pesquisa de novas estratégias terapêuticas

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014Classic galactosemia is a potentially lethal disease of the galactose metabolism caused by a severe deficiency of GALT, the second enzyme of the Leloir pathway. This disorder is associated with mutations in the GALT gene, and displays an autosomal recessive pattern of inheritance. Although apparently asymptomatic at birth, affected infants start developing escalating symptoms after 1 to 2 weeks of galactose ingestion due to milk feeding. Upon implementation of a galactose-restricted diet, infants show rapid clinical improvement and seem to be almost miraculously cured; however, the long-term outcome is often disappointing, as most patients go on to develop severe complications notwithstanding strict compliance with the dietary therapy. Despite decades of intensive research, classic galactosemia pathophysiology is still largely unknown, resulting in a limited and poorly effective therapy. A comprehensive understanding of the molecular mechanisms underlying classic galactosemia potentially opens new therapeutic avenues, and prompted us to develop the present work. The first part of this thesis (chapter 1) comprises a general introduction with a review of the literature concerning the molecular and biochemical basis of classic galactosemia, followed by an overview of the current therapeutic approaches in genetic disorders – with a special focus on inherited metabolic disorders – and finally the objectives of the present work. Our studies initiated with the molecular characterization of all patients currently followed by the Portuguese metabolic centers (chapter 2). The rationale for this study was the previous description that GALT genotype represents a valuable prognostic tool for the outcome of galactosemic patients. Accordingly, after genotyping 42 Portuguese galactosemic patients, we searched for genotype-phenotype correlations by a 3-fold evaluation. Firstly, we employed in silico strategies to assess the structural-functional impact of previously uncharacterized mutations; secondly, we evaluated the biochemical phenotype at both the metabolite and enzymatic levels, represented by Gal-1-P values and GALT activity in erythrocytes; and finally, evaluated the resulting clinical outcome. Establishment of correlations between genotype and biochemical or clinical phenotypes, however, was poorly attained, reiterating the complexity of this disease and emphasizing the idea that other modifiers – possibly genetic, epigenetic and environmental – contribute to the pathophysiology of classic galactosemia. The update of the Portuguese GALT mutational spectrum revealed the intronic mutation c.820+13A>G (IVS8+13A>G) as the second most prevalent variation, strongly suggesting its pathogenicity, which set the basis for the study described in chapter 3. We functionally characterized the c.820+13A>G variation by ex vivo and in vivo analyses, which were in full agreement with the previous in silico predictions. Indeed, we confirmed this variation is a disease-causing mutation, whose mechanism of action involves the activation of a cryptic donor site, which, in turn, induces an aberrant splicing of the GALT pre-mRNA, thus causing a frameshift with inclusion of a premature stop codon. Structural-functional studies of the recombinant truncated GALT showed it was devoid of enzymatic activity and prone to aggregation. Finally, antisense oligonucleotides were designed to specifically recognize the mutation, and successfully restored the constitutive splicing. Molecular studies in several inherited metabolic disorders have led to the realization that only a minor part of the mutations directly disrupt functional sites of the proteins. Accordingly, in chapter 4, we described the structural and functional characterization of the most prevalent mutations in the GALT gene – p.Q188R, p.S135L, p.K285N and p.N314D – and of other five clinically relevant mutations – p.R148Q, p.G175D, p.P185S, p.R231C and p.R231H. Interestingly, the analyzed mutations did not affect the global conformational stability of the GALT enzyme; rather, most mutations, notwithstanding their impact on the enzyme functionality, increased the propensity for aggregation, which at the cellular level reflects in a decrease of the enzyme’s effective concentration. These results are in agreement with previous studies in classic galactosemia models, and suggest that GALT aggregation might be a major pathogenic mechanism underlying this disorder. Previous studies have reported a yeast galactosemia model allowing the evaluation of human GALT mutations severity, by assaying the sensitivity of transformed yeast cultures to galactose addition to the medium. Thus, in chapter 5, we developed a prokaryotic model of galactose sensitivity to evaluate the ability of the above referred human GALT mutants in alleviating the galactose-induced toxicity. This model presents the inherent advantage of being assayed in vivo, thus providing valuable insights on mutations’ impact on human GALT function. Additionally, arginine ability to ameliorate the galactose-induced toxicity was also evaluated for each human GALT mutant. The rationale for this approach was, not only the long-recognized anti-aggregation properties of arginine, but also its important therapeutic effect described in previous studies. In effect, arginine appears to exert a mutation-specific mode of action, alleviating the galactose toxicity in the p.Q188R, p.K285N, and p.G175D mutants, which suggests that might be of some benefit in classic galactosemia. Nonetheless, further studies are underway to ascertain arginine’s potential therapeutic effect in this inherited disorder of the galactose metabolism. Chapter 6 presents a general discussion and major conclusions disclosed by this work, framing them in the current state of the art, proving also some perspectives for future studies. Taken together, our results provide important insights on classic galactosemia, namely by shedding light on the underlying pathogenic molecular mechanisms, thus contributing for a better understanding of this enigmatic disorder. Finally, and importantly, these studies paved the way to the search, development and improvement of novel and alternative therapeutic strategies, so needed to overcome the overwhelming and burdensome long-term complications presented by most classic galactosemic patients.Após a sua internalização celular, a galactose é rapidamente metabolizada a glucose-1- fosfato pela acção sequencial de três enzimas: galactocinase (GALK), galactose-1-fosfato uridililtransferase (GALT) e uridina difosfogalactose 4’-epimerase (GALE). Estas enzimas permitem, respectivamente, a fosforilação da galactose em galactose-1-fosfato (GALK), a conversão de galactose-1-fosfato e uridina difosfoglucose em glucose-1-fosfato e uridina difosfogalactose (GALT), e a interconversão de uridina difosfoglucose em uridina difosfogalactose (GALE). Estas enzimas constituem a via de Leloir e, apesar de o fígado ser o principal órgão envolvido no metabolismo da galactose, encontram-se na maioria das células e dos tecidos. Uma deficiência enzimática em qualquer uma das três enzimas da via de Leloir resulta numa diminuição ou ausência de capacidade de metabolizar a galactose e, consequentemente, conduz à sua acumulação no sangue – hipergalactosémia. A Galactosémia Clássica (OMIM #230400), a forma mais comum de hipergalactosémia primária, é uma doença genética de transmissão autossómica recessiva, que afecta 1 em cada 30.000 a 60.000 nados-vivos, apresentando uma prevalência variável entre populações, nomeadamente na Irlanda e Turquia onde atinge valores de 1/23.500 - 1/23.775 e no Japão onde a sua prevalência atinge o valor mais baixo (1/1.000.000). A ausência ou diminuição de actividade da GALT é causada por mutações no gene GALT, e encontram-se descritas mais de 250 variações, reflectindo a elevada heterogeneidade alélica desta doença metabólica. Para além disso, a maioria dos doentes são heterozigóticos compostos, um dos factores determinantes da ampla variabilidade fenotípica observada. Aquando do nascimento, a criança aparenta ser assintomática. Os sintomas tornam-se evidentes após o início da ingestão de leite, e consistem inicialmente em dificuldades alimentares e de desenvolvimento, vómitos, diarreia, letargia e hipotonia, podendo evoluir para cataratas e septicémia, e eventualmente conduzir à morte. Após implementação de uma dieta restrita em galactose – o tratamento padrão – as crianças mostram notáveis melhorias em apenas 24 horas, e em apenas uma a duas semanas as disfunções hepática e renal desaparecem completamente. Esta resposta dramática à terapia dietética levou ao conceito da Galactosémia Clássica como uma doença relativamente benigna e fácil de tratar. No entanto, os doentes com Galactosémia Clássica desenvolvem complicações a longo prazo e a diversos níveis, como neurológico e psiconeurológico, crescimento e densidade óssea, e disfunção ovárica nas mulheres, os quais parecem ser independentes de um diagnóstico precoce e de uma adesão do doente à terapia. Apesar de a primeira descrição datar de 1908, a Galactosémia Clássica continua a ser um enigma, quer ao nível do conhecimento aprofundado da sua fisiopatologia, quer ao nível do desenvolvimento de alternativas terapêuticas que permitam, sobretudo, mitigar as complicações a longo prazo. O presente trabalho pretende, assim, contribuir para a elucidação dos mecanismos moleculares subjacentes à patogénese das mutações GALT prevalentes na população galactosémica Portuguesa, assim como explorar novas estratégias terapêuticas direccionadas para cada tipo específico de mutação. O primeiro capítulo da tese apresenta uma revisão geral da literatura sobre vários aspectos relacionados com a Galactosémia Clássica: as bases bioquímicas e moleculares, a fisiopatologia e as actuais abordagens terapêuticas. Em seguida, descrevem-se em pormenor os mecanismos moleculares subjacentes à maioria das mutações que originam Erros Hereditários do Metabolismo (onde se inclui a Galactosémia Clássica) e que causam perca de função, nomeadamente as mutações missense e as que afectam o splicing, mecanismos esses que condicionam as novas terapias desenhadas de acordo com o tipo de mutação. Por fim, são descritos os objectivos delineados para o presente trabalho. O trabalho experimental teve início com a caracterização molecular de todos os doentes actualmente seguidos nos centros metabólicos nacionais de Lisboa, Porto e Coimbra, sendo o objectivo final averiguar se, como sugerido na literatura, o genótipo GALT pode ter um valor prognóstico sobre a evolução fenotípica dos doentes galactosémicos (capítulo 2). Os dados obtidos permitiram estabelecer o espectro mutacional da Galactosémia Clássica em Portugal e compará-lo com o de outras populações. O estudo de 42 doentes revelou a presença de 14 substituições nucleotídicas, sendo 10 missense, 2 nonsense e 2 de splicing. Identificaram-se 16 genótipos diferentes, mas metade dos doentes são homozigóticos para p.Q188R, a mutação prevalente não só em Portugal como a nível mundial. Surpreendentemente, a segunda mutação mais frequente é uma mutação de splicing, descrita até então como benigna. Em seguida, e recorrendo a programas bioinformáticos adequados, procedeu-se à análise da potencial patogenicidade das mutações ainda não caracterizadas na literatura. Os resultados sugeriram que a maioria destas mutações missense afectará a estabilidade e a funcionalidade da proteína mutada, enquanto que a mutação de splicing mais frequente deverá induzir um mecanismo de splicing alternativo. Por fim, este estudo revelou, na maioria dos casos, a ausência de uma correlação clara entre a gravidade das mutações prevista pela análise in silico e o fenótipo bioquímico dos doentes, determinado pelos níveis eritrocitários de galactose-1-fosfato, assim como com o fenótipo clínico e a evolução desfavorável manifestada pelos doentes. No entanto, tal resultado não surpreende dado a Galactosémia Clássica, apesar de ser uma doença monogénica, não originar fenótipos claros. Este facto prende-se com a falta de informação estrutural sobre os componentes enzimáticos da via de Leloir, colocando-se a hipótese de uma alteração na GALT poder afectar toda a via metabólica. Por outro lado, os metabolitos da galactose estão envolvidos em diversas reacções fisiológicas, nomeadamente as de glicosilação que se reflectem aos mais variados níveis. Finalmente, a influência de outros genes modificadores, de alterações epigenéticas e de factores ambientais também não pode ser ignorada. Os capítulos seguintes são dedicados ao estudo dos mecanismos moleculares subjacentes às mutações prevalentes na população Portuguesa e ao desenvolvimento de novas abordagens terapêuticas. No capítulo 3 descreve-se o estudo da mutação de splicing IVS8+13A>G, até então considerada como benigna, mas que revelou ser a segunda mais frequente na nossa população galactosémica. A caracterização funcional do mecanismo patogénico foi efectuada por transfeção de um minigene contendo a sequência mutada em duas linhas diferentes de células eucarióticas. A análise dos produtos de transcrição revelou que a mutação activa um sítio críptico de splicing, causando um splicing anómalo do pré-mRNA GALT, o qual induz um frameshift com inclusão de um codão de terminação prematuro (p.D274GfsX291). Por outro lado, dado ter-se observado a presença do mensageiro mutado nas amostras biológicas dos doentes portadores desta mutação, colocou-se a hipótese de a proteína truncada ser produzida in vivo, o que levou à produção da proteína recombinante. Os estudos estruturais e funcionais subsequentes revelaram que esta proteína é propensa à formação de agregados e é destituída de actividade enzimática. Finalmente, e utilizando oligonucleótidos antisense que hibridam especificamente com o local da mutação, impedindo assim a ligação do spliceossoma e forçando a sua ligação ao sítio canónico dador de splicing, foi possível corrigir o splicing alternativo induzido pela mutação e obter o mensageiro selvagem. Estas experiências constituíram a prova de conceito sobre a aplicabilidade da terapia antisense como alternativa estratégica para a claramente insuficiente dieta restrita em galactose. ! O espectro mutacional da maioria dos erros hereditários do metabolismo é dominado por mutações do tipo missense. O capítulo 4 aborda a caracterização estrutural e funcional das quatro variantes GALT prevalentes a nível mundial, assim como outras cinco variantes com relevância clínica, nomeadamente na população galactosémica Portuguesa. Diversas metodologias foram empregues para determinação da actividade enzimática e do perfil de inactivação térmica, bem como métodos biofísicos para avaliação das estruturas secundária, terciária e quaternária das proteínas mutadas. Os resultados revelaram que as mutações pontuais não afectam nenhuma das estruturas acima mencionadas, mas sim a propensão para uma agregação precoce destas variantes. Este resultado constitui na realidade a principal novidade, dado que um estudo recente postulou que algumas mutações no gene GALT afectam o correcto folding das proteínas mutadas. Esta conclusão é extremamente relevante na medida em que, para além da diminuição da actividade uridililtransferásica, a acumulação de agregados proteicos interfere com a homeostase celular. Efectivamente, diversos estudos relataram a presença, em doentes galactosémicos, do aumento de actividade dos sistemas ligados ao stress do retículo endoplasmático e da unfolded protein response, assim como de níveis elevados de stress oxidativo, fenómenos característico de uma proteotoxicidade. Estes dados levam-nos então a colocar como potencial e nova hipótese terapêutica a utilização de moduladores da proteostase, os quais prolongam a semi-vida celular das variantes GALT, compensando assim parcialmente a diminuição de actividade enzimática e simultaneamente prevenindo a acumulação de agregados proteicos. No capítulo 5 desenvolvemos um modelo procariótico de sensibilidade à galactose que permitisse avaliar, não só o impacto causado pelas diversas mutações missense, mas também o efeito de compostos com potencial acção terapêutica. Utilizámos uma estirpe de E. coli com o gene galT endógeno deletado, de modo que toda a actividade GALT detectada é proveniente do mutante nela transformado. Os resultados, embora ainda preliminares, confirmaram a validade do modelo delineado e permitiram obter dados a dois níveis. Por um lado, replicámos in vivo os resultados previamente obtidos in vitro quanto à capacidade de aliviar a toxicidade induzida pela galactose, por comparação com a enzima selvagem. Por outro lado, e muito relevante, verificámos que o modelo é válido para testar moléculas com potencial terapêutico. Efectivamente, algumas destas enzimas mutadas revelaram-se sensíveis à arginina, um composto amplamente reconhecido como estabilizador de proteínas e anti-agregante, aliviando desse modo a toxicidade da galactose para a bactéria. A presente tese termina com o capítulo 6 no qual se apresenta uma discussão dos resultados obtidos, incluindo uma análise integrada de todo o trabalho e respectivas conclusões, bem como algumas perspectivas de trabalho futuro. Em suma, este trabalho contribuiu para um melhor conhecimento da Galactosémia Clássica em Portugal e para a elucidação dos mecanismos patogénicos subjacentes às mutações prevalentes, bem como para a descoberta e desenvolvimento de novas alternativas terapêuticas, tão necessárias para minorar as graves sequelas que a maioria dos doentes galactosémicos apresenta a longo termo.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/48259/2008, projeto PEst-OE/SAU/UI4013/2011