Intrdodução: As terapias com anticonvulsivantes de anel aromático (ACA) são freqüentemente associadas a reações adversas. No entanto, reações de hipersensibilidade (RH) não-imediatas (tardias) a estes fármacos são raras, imprevisíveis e geralmente relacionadas à alta morbidade e mortalidade. Foi demonstrado que estas RH aos ACA estão fortemente associadas ao Antígenio de Leucócitos Humanos (HLA)-B*1502 em pacientes chineses e ao HLA-A*3101 em caucasianos. Polimorfismos de genes do metabolismo do Citocromo P450 (CYP)2C9 foram mais associados a estas reações em pacientes orientais. Objetivo: Nosso objetivo é analisar a associação das reações de hipersensibilidade a anticonvulsivantes de anel aromático com os polimorfismos descritos e de interesse, bem como realizar a tipificação de HLA em uma população de São Paulo, Brasil. Métodos: Estudo tipo caso-controle com genotipagem dos polimorfismos de interesse por reação em cadeia da polimerase (PCR) em tempo real e tificação de HLA A, B, C, DRB, DQA, DQB por PCR seguido de deteção utilizando método LuminexR. A avaliação fenotípica se baseou em sistemas de escores padronizados, utilizando um questionário adaptado da ENDA (Rede Européia de Alergia a Medicamentos), em registros médicos e no acompanhamento clínico. O teste de contato com o medicamento suspeito foi realizado de acordo com as recomendações da ENDA, nos pacientes que apresentaram reação. Resultados: Foram estudados 506 pacientes, 65% do gênero feminino e a idade média foi de 43,6 anos. Oitenta por cento era de etnia mista. Polimorfismos de HLA-A*3101, HLA-B*1502, CYP2C9, CYP2C19 e CYP3A5 foram analisados de 55 indivíduos com reações de hipersensibilidade (RH) a antiepilépticos, de 85 tolerantes e de 366 controles sadios. Dos 55 casos foram validados como RH, 32 apresentaram Reação a Drogas com eosinofilia e sintomas sistêmicos (DRESS), 12 Síndrome de Stevens-Johnson (SSJ) e 11 exantema maculo-papular. De todos os 46 testes de contato com medicamento, 29 (63%) foram positivos, tanto em SSJ como em DRESS. Houve associação significativa entre polimorfismo de HLA-A*1502 e casos. Nenhum de nossos grupos de estudo apresentou associação positiva com polimorfismos de HLAA* 3101. Verificamos uma forte associação entre a atividade normal do CYP3A5 e indivíduos tolerantes quando comparado com casos (p = 0,0002, OR = 4,8). A tipificação de HLA demonstrou associação significante de HLA-A*31, HLA-A*74, HLA-B*35 e HLA-B*53 com reações graves aos ACA e de HLA-B*44 e HLA-C*03 com indivíduos tolerantes. Conclusão: Estes resultados sugere fortemente a existência de fatores genéticos de risco e/ou de proteção a RH a ACA em indivíduos brasileiros, mas não devem ser considerados de forma isolada. Assim, a relevância deste estudo extrapola o objetivo de estudo caso-controle e sugere um modelo como forma de prevenção primária às RH aos ACA. / Background: Antiepileptics with aromatic ring (AAR) therapies are frequently associated with adverse reactions. Nevertheless non-immediate (late) hypersensitivity reactions (HR) to these drugs are rare, unpredictable and usually related with high morbidity and mortality. A strong pharmacogenetic association has been reported in Chinese patients with these HR and Human Leukocyte Antigen (HLA)-B*1502 and with HLA- A*3101 in caucasians. Polymorphism of genes of P450 Cytocrome (CYP)2C9 has been related to these reactions in patients of oriental origin. Objective: Our aim is to analyze the association between hypersensitivity reactions due to AAR and the described polymorphisms, as well as perform the typification of HLA in a population of São Paulo, Brazil. Methods: Case-control study genotyping the polymorphisms of interest by polymerase chain reaction (PCR) real time and typifying HLA A, B, C, DRB, DQA, DQB by PCR followed by LuminexR .The phenotype evaluation was based on standardized scoring systems using an adapted ENDA (European Network of Drug Allergy) questionnaire, medical records and on the clinical follow-up in our Allergy Clinic. The patch test with the culprit drug was performed in patients who experienced HR according to the ENDA recommendations. Results: We studied 506 subjects, 65% female and mean age was 43,6 years. Eighty percent had mixed ethnicity. Polymorphisms of HLA-B*1502, HLA- A*3101, CYP2C9, CYP2C19 e CYP3A5 were studied in 55 subjects with antiepileptics HR, 85 tolerants, and 366 control subjects. Of 55 cases were validated as AHR, 32 presented Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS), 12 Stevens-Johnson Syndrome (SJS) and 11 maculopapular exanthema. Of all 46 drug patch tests, 29 (63%) were positive, in both SJS and DRESS. A significant association between polymorphism of HLA-A*1502 and cases was found. None of our study groups presented positive association with HLA-A*3101 polymorphisms. We found a strong association between the normal activity of CYP3A5 and tolerants subjects when compared to HR (p=0.0002, OR=4.8). The HLA typification showed a significant association between HLA-A*31, HLA-A*74, HLAB* 35 e HLA-B*53 and severe AAR reactions and HLA-B*44 and HLA-C*03 in tolerants subjects. Conclusion: These results strongly suggests the existence of genetic risk and/or protective factors to the development of HR to AAR AAR in Brazilian subjects, but it should not be considered in a isolated manner. So, the relevance of this study extrapolates the aim of a case-control study and suggests a system of primary prevention to HR due to AAR

Cette thèse comporte quatre fichiers vidéo. This thesis comes with four video files. / L'industrie chimique mondiale est en pleine mutation, cherchant des solutions pour rendre la synthèse organique classique plus durable. Une telle solution consiste à passer de la catalyse chimique classique à la biocatalyse. Bien que les avantages des enzymes incluent leur stéréo, régio et chimiosélectivité, cette sélectivité réduit souvent leur promiscuité. Les efforts requis pour adapter la fonction enzymatique aux réactions désirées se sont révélés d'une efficacité modérée, de sorte que des méthodes rapides et rentables sont nécessaires pour générer des biocatalyseurs qui rendront la production chimique plus efficace. Dans l’ère de la bioinformatique et des outils de calcul pour soutenir l'ingénierie des enzymes, le développement rapide de nouvelles fonctions enzymatiques devient une réalité. Cette thèse commence par un examen des développements récents sur les outils de calcul pour l’ingénierie des enzymes. Ceci est suivi par un exemple de l’ingénierie des enzymes purement expérimental ainsi que de l’évolution des protéines. Nous avons exploré l’espace mutationnel d'une enzyme primitive, la dihydrofolate réductase R67 (DHFR R67), en utilisant l’ingénierie semi-rationnelle des protéines. La conception rationnelle d’une librarie de mutants, ou «Smart library design», impliquait l’association covalente de monomères de l’homotétramère DHFR R67 en dimères afin d’augmenter la diversité de la librairie d’enzymes mutées. Le criblage par activité enzymatique a révélé un fort biais pour le maintien de la séquence native dans un des protomères tout en tolérant une variation de séquence élevée pour le deuxième. Il est plausible que les protomères natifs procurent l’activité observée, de sorte que nos efforts pour modifier le site actif de la DHFR R67 peuvent n’avoir été que modérément fructueux. Les limites des méthodes expérimentales sont ensuite abordées par le développement d’outils qui facilitent la prédiction des points chauds mutationnels, c’est-à-dire les sites privilégiés à muter afin de moduler la fonction. Le développement de ces techniques est intensif en termes de calcul, car les protéines sont de grandes molécules complexes dans un environnement à base d’eau, l’un des solvants les plus difficiles à modéliser. Nous présentons l’identification rapide des points chauds mutationnels spécifiques au substrat en utilisant l'exemple d’une enzyme cytochrome P450 industriellement pertinente, la CYP102A1. En appliquant la technique de simulation de la dynamique moléculaire par la force de polarisation adaptative, ou «ABF», nous confirmons les points chauds mutationnels connus pour l’hydroxylation des acides gras tout en identifiant de nouveaux points chauds mutationnels. Nous prédisons également la conformation du substrat naturel, l’acide palmitique, dans le site actif et nous appliquons ces connaissances pour effectuer un criblage virtuel d'autres substrats de cette enzyme. Nous effectuons ensuite des simulations de dynamique moléculaire pour traiter l’impact potentiel de la dynamique des protéines sur la catalyse enzymatique, qui est le sujet de discussions animées entre les experts du domaine. Avec la disponibilité accrue de structures cristallines dans la banque de données de protéines (PDB), il devient clair qu’une seule structure de protéine n’est pas suffisante pour élucider la fonction enzymatique. Nous le démontrons en analysant quatre structures cristallines que nous avons obtenues d’une enzyme β-lactamase, parmi lesquelles un réarrangement important des résidus clés du site actif est observable. Nous avons réalisé de longues simulations de dynamique moléculaire pour générer un ensemble de structures suggérant que les structures cristallines ne reflètent pas nécessairement la conformation de plus basse énergie. Enfin, nous étudions la nécessité de compléter de manière informatisée un hémisphère où l’expérimental n’est actuellement pas possible, à savoir la prédiction de la migration des gaz dans les enzymes. À titre d'exemple, la réactivité des enzymes cytochrome P450 dépend de la disponibilité des molécules d’oxygène envers l’hème du site actif. Par le biais de simulations de la dynamique moléculaire de type Simulation Implicite du Ligand (ILS), nous dérivons le paysage de l’énergie libre de petites molécules neutres de gaz pour cartographier les canaux potentiels empruntés par les gaz dans les cytochromes P450 : CYP102A1 et CYP102A5. La comparaison pour les gaz CO, N2 et O2 suggère que ces enzymes évoluent vers l’exclusion du CO inhibiteur. De plus, nous prédisons que les canaux empruntés par les gaz sont distincts des canaux empruntés par le substrat connu et que ces canaux peuvent donc être modifiés indépendamment les uns des autres. / The chemical industry worldwide is at a turning point, seeking solutions to make classical organic synthesis more sustainable. One such solution is to shift from classical catalysis to biocatalysis. Although the advantages of enzymes include their stereo-, regio-, and chemoselectivity, their selectivity often reduces versatility. Past efforts to tailor enzymatic function towards desired reactions have met with moderate effectiveness, such that fast and cost-effective methods are in demand to generate biocatalysts that will render the production of fine and bulk chemical production more benign. In the wake of bioinformatics and computational tools to support enzyme engineering, the fast development of new enzyme functions is becoming a reality. This thesis begins with a review of recent developments on computational tools for enzyme engineering. This is followed by an example of purely experimental enzyme engineering and protein evolution. We explored the mutational space of a primitive enzyme, the R67 dihydrofolate reductase (DHFR), using semi-rational protein engineering. ‘Smart library design’ involved fusing monomers of the homotetrameric R67 DHFR into dimers, to increase the diversity in the resulting mutated enzyme libraries. Activity-based screening revealed a strong bias for maintenance of the native sequence in one protomer with tolerance for high sequence variation in the second. It is plausible that the native protomers procure the observed activity, such that our efforts to modify the enzyme active site may have been only moderately fruitful. The limitations of experimental methods are then addressed by developing tools that facilitate computational mutational hotspot prediction. Developing these techniques is computationally intensive, as proteins are large molecular objects and work in aqueous media, one of the most complex solvents to model. We present the rapid, substrate-specific identification of mutational hotspots using the example of the industrially relevant P450 cytochrome CYP102A1. Applying the adaptive biasing force (ABF) molecular dynamics simulation technique, we confirm the known mutational hotspots for fatty acid hydroxylation and identify a new one. We also predict a catalytic binding pose for the natural substrate, palmitic acid, and apply that knowledge to perform virtual screening for further substrates for this enzyme. We then perform molecular dynamics simulations to address the potential impact of protein dynamics on enzyme catalysis, which is the topic of heated discussions among experts in the field. With the availability of more crystal structures in the Protein Data Bank, it is becoming clear that a single protein structure is not sufficient to elucidate enzyme function. We demonstrate this by analyzing four crystal structures we obtained of a β-lactamase enzyme, among which a striking rearrangement of key active site residues was observed. We performed long molecular dynamics simulations to generate a structural ensemble that suggests that crystal structures do not necessarily reflect the conformation of lowest energy. Finally, we address the need to computationally complement an area where experimentation is not currently possible, namely the prediction of gas migration into enzymes. As an example, the reactivity of P450 cytochrome enzymes depends on the availability of molecular oxygen at the active-site heme. Using the Implicit Ligand Sampling (ILS) molecular dynamics simulation technique, we derive the free energy landscape of small neutral gas molecules to map potential gas channels in cytochrome P450 CYP102A1 and CYP102A5. Comparison of CO, N2 and O2 suggests that those enzymes evolved towards exclusion of the inhibiting CO. In addition, we predict that gas channels are distinct from known substrate channels and therefore can be engineered independently from one another.

Indiana University-Purdue University Indianapolis (IUPUI) / Drug-drug interactions (DDIs) refer to situations where one drug affects the pharmacokinetics or pharmacodynamics of another. DDIs represent a major cause of morbidity and mortality. A common adverse drug reaction (ADR) that can result from, or be exacerbated by DDIs is drug-induced myopathy. Identifying DDIs and understanding their underlying mechanisms is key to the prevention of undesirable effects of DDIs and to efforts to optimize therapeutic outcomes. This dissertation is dedicated to identification of clinically important myopathic DDIs and to elucidation of their underlying mechanisms. Using data mined from the published cytochrome P450 (CYP) drug interaction literature, 13,197 drug pairs were predicted to potentially interact by pairing a substrate and an inhibitor of a major CYP isoform in humans. Prescribing data for these drug pairs and their associations with myopathy were then examined in a large electronic medical record database. The analyses identified fifteen drug pairs as DDIs significantly associated with an increased risk of myopathy. These significant myopathic DDIs involved clinically important drugs including alprazolam, chloroquine, duloxetine, hydroxychloroquine, loratadine, omeprazole, promethazine, quetiapine, risperidone, ropinirole, trazodone and simvastatin. Data from in vitro experiments indicated that the interaction between quetiapine and chloroquine (risk ratio, RR, 2.17, p-value 5.29E-05) may result from the inhibitory effects of quetiapine on chloroquine metabolism by cytochrome P450s (CYPs). The in vitro data also suggested that the interaction between simvastatin and loratadine (RR 1.6, p-value 4.75E-07) may result from synergistic toxicity of simvastatin and desloratadine, the major metabolite of loratadine, to muscle cells, and from the inhibitory effect of simvastatin acid, the active metabolite of simvastatin, on the hepatic uptake of desloratadine via OATP1B1/1B3. Our data not only identified unknown myopathic DDIs of clinical consequence, but also shed light on their underlying pharmacokinetic and pharmacodynamic mechanisms. More importantly, our approach exemplified a new strategy for identification and investigation of DDIs, one that combined literature mining using bioinformatic algorithms, ADR detection using a pharmacoepidemiologic design, and mechanistic studies employing in vitro experimental models.

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Die Therapie der HIV Infektion besteht aus Kombination mehrerer antiretroviraler Substanzen und birgt ein erhöhtes Risiko an Arzneimittelwechselwirkungen. Das bekannte Problem der Virusresistenz kann zudem durch Enzyminduktion begünstigt werden. Das Ziel der vorliegenden Arbeit lag in Untersuchungen zu Einflüssen der Virustatika auf die Expression von Cytochrom P450 Enzymen: 1A1, 1B1, 3A4 sowie der P-Glykoproteins (MDR1) an immortalisierten Zellsystemen. Die Protease Inhibitoren Indinavir, Nelfinavir, Ritonavir und Saquinavir induzierten die Regulation der mRNA Expression über den Aryl-Kohlenwasserstoff-Rezeptor (AhR) und den Pregnan-X-Rezeptor (PXR) dosisabhängig und signifikant. Die Nukleosidischen Reverse Transkriptase Inhibitoren Zalcitabin, Zidovudin und Lamivudin sowie der Nicht-Nukleosidische Inhibitor Nevirapin zeigten induktive Eigenschaften nur für die AhR Zielgene CYP1A1 und CYP1B1. Amprenavir und Efavirenz aktivierten die PXR-Regulation. Die möglichen Auswirkungen der Induktion der untersuchten Gene wurden ausführlich diskutiert. Die molekularen Grundlagen der interindividuell variierenden Aktivität von CYP3A wurden in einer Probandenstudie untersucht. Es wurden die mRNA Expression in den Leukozyten, die Aktivität des Enzyms und einige bekannte Polymorphismen unter Einwirkung von Rifampicin untersucht und diskutiert. / The therapy of HIV infection requires a combination of several antiretroviral substances accompanying risk factors for drug-drug interactions. Moreover, virus resistance can be promoted by enzyme induction caused by antiretroviral drugs. The aim of the study was to investigate the influences of antiretroviral substances on the expression of cytochrome P450 enzymes: 1A1, 1B1, 3A4 and p-glycoprotein (MDR1) using immortalized cell systems. The protease inhibitors indinavir, nelfinavir, ritonavir and saquinavir induced significantly the regulation of mRNA expression through the aryl hydrocarbon receptor (AhR) and the pregnane-x-receptor (PXR) in a concentration-dependent manner. The nucleoside reverse transcriptase inhibitors zalcitabine, zidovudine and lamivudine and the non-nucleoside reverse transcriptase inhibitor nevirapine showed inductive properties only for the AhR target genes CYP1A1 and CYP1B1.Amprenavir and efavirenz activated the PXR target genes. Potentially effects of the described induction are discussed. In a second part of the work, the molecular mechanisms of the individual varying activity of the CYP3A enzyme were investigated applying an in vivo study. CYP3A4 mRNA expression and rifampicin mediated induction in leucocytes were correlated with systemic enzyme activity under induction and known polymorphisms.