Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-03T22:31:54Z No. of bitstreams: 1 JessicaDeFatimaVianna_DISSERT.pdf: 3724208 bytes, checksum: f7d62fbcd54bf6b212f2003b461810c5 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-11T18:14:29Z (GMT) No. of bitstreams: 1 JessicaDeFatimaVianna_DISSERT.pdf: 3724208 bytes, checksum: f7d62fbcd54bf6b212f2003b461810c5 (MD5) / Made available in DSpace on 2017-04-11T18:14:29Z (GMT). No. of bitstreams: 1 JessicaDeFatimaVianna_DISSERT.pdf: 3724208 bytes, checksum: f7d62fbcd54bf6b212f2003b461810c5 (MD5) Previous issue date: 2017-02-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A tuberculose ? uma doen?a bacteriana provocada pelo Mycobacterium tuberculosis, e de acordo com a Organiza??o Mundial de Sa?de, apenas em 2015 foram 10,4 milh?es de novos casos relatados e 1,4 milh?o de mortes. Cresce o n?mero de casos de pacientes infectados com cepas resistentes aos antimicrobianos mais comumente utilizados, fazendo-se necess?rio uso de drogas de segunda-linha. A capreomicina e a estreptomicina encaixam-se nesse grupo, e s?o antibi?ticos que possuem como mecanismo de atua??o a inibi??o da s?ntese proteica. Entretanto, seus mecanismos de liga??o em seus s?tios s?o distintos: a capreomicina ? capaz de se ligar a ambas subunidades ribossomais (30S e 50S), enquanto que a estreptomicina liga-se ? subunidade ribossomal menor (30S), e interage com alguns pontos da prote?na S12. Atrav?s de dados cristalogr?ficos e simula??es computacionais, foi calculada a energia de intera??o da capreomicina e da estreptomicina com cada um dos res?duos constituintes de seus s?tios utilizando a Teoria Funcional da Densidade (DFT) e do M?todo de Fracionamento Molecular com Capas Conjugadas (MFCC). Os resultados revelaram valores energ?ticos de cada nucleot?deo pertencente ao s?tio de liga??o desses dois medicamentos, como tamb?m dos amino?cidos da prote?na S12 com os quais a estreptomicina interage. Assim, para a capreomicina na subunidade 30S, foram avaliados res?duos presentes em um raio de at? 14 ? distantes do f?rmaco, totalizando 44 res?duos; e na subunidade 50S, 30 nucleot?deos foram analisados, e estavam distribu?dos at? o raio de 30 ? de dist?ncia. Com a estreptomicina foram levados em considera??o 60 nucleot?deos distribu?dos at? 12,5 ? de dist?ncia da droga na subunidade 30S, e 25 amino?cidos da prote?na S12 com at? 15 ? de dist?ncia. Identificamos tamb?m as contribui??es das liga??es de hidrog?nio e das intera??es hidrof?bicas nas intera??es f?rmaco-receptor; as regi?es dos f?rmacos que mais contribu?ram para as fixa??es desses em seus s?tios de liga??o; como tamb?m a identifica??o dos res?duos que s?o mais associados ?s muta??es e consequente resist?ncia. / Tuberculosis is a disease caused by Mycobacterium tuberculosis, and according to the World Health Organization, only in 2015 occurred 10.4 million new cases reported and 1.4 million deaths. The number of cases of patients infected with antimicrobial resistant strains most used is increasing, requiring the use of second-line drugs. Capreomycin and streptomycin are part of the group, and are antibiotics whose mechanism of action is the inhibition of protein synthesis. However, its binding mechanisms in their sites are distinct: capreomycin is able to bind to both ribosomal (30S and 50S) subunits, whereas streptomycin binds to the smaller ribosomal subunit (30S), and interacts with some points of S12 protein. Through crystallographic data and computational simulations, we calculated the interaction energy of capreomycin and streptomycin with each of the residues component of their sites using the Density Functional Theory (DFT) and Molecular Fractionation with Conjugated Caps (MFCC). The results showed energy values of each nucleotide belonging to binding site of these two drugs, as well as the amino acids of the S12 protein with which streptomycin interacts. Thus, for capreomycin in the 30S subunit, residues present in a radius of up to 14 ? distant from the drug, totaling 44 residues; and in the 50S subunit, 30 nucleotides were analyzed, and were distributed up to the 30? radius distance. Regarding streptomycin, 60 nucleotides distributed up to 12.5 ? away from the drug in the 30S subunit, and 25 amino acids of the S12 protein with up to 15 ? were taken into account. We also identify the contributions of hydrogen bonds and hydrophobic interactions in drug-receptor interactions; the regions of the drugs that most contributed to the anchorages of these in their binding sites; as well as the identification of residues that are most associated with mutations and consequent resistance.

La tuberculosis es una enfermedad muy prevalente en el Perú y los casos de tuberculosis multidrogorresistente (MDR) están en aumento. La capreomicina es un antibiótico péptido cíclico utilizado anteriormente en el esquema de segunda línea de tratamiento para tuberculosis MDR. El mecanismo molecular de acción conocido de este fármaco es la inhibición de la fase de elongación de proteínas. En esta investigación utilizamos el microorganismo E. coli, por ser un gold standard para estudios mecanísticos de antibióticos y un espectofotómetro , llamado Stopped-flow, para evidenciar los cambios conformaciones del factor de iniciación 3 fluorescente (IF3DL). Se observó que la unión de capreomicina a la subunidad menor 30S del ribosoma promueve el acercamiento de los dominios de IF3DL, el cual fue evidenciado como una disminución de fluorescencia. Esta reacción fue dependiente de concentración, encontrándose una mayor disminución de la fluorescencia en función del aumento de capreomicina. Cuando se añadió el factor de iniciación 1 (IF1) al complejo 30S-IF3DL, la unión de capreomicina también resultó en un acercamiento de los dominios de IF3DL, pero ligeramente menor que la que ocurre en ausencia de IF1. Los parámetros cinéticos entre los complejos 30S – IF3DL en ausencia o presencia de IF1 con capreomicina indican que el acercamiento de los dominios de IF3DL es independiente de IF1. En conclusión, capreomicina induce un acercamiento de los dominios de IF3, lo que podría afectar su función. / Tuberculosis is a prevalent disease in Peru and cases of multridrugresistant tuberculosis (MDR – TB) are increasing. Capreomycin is a ciclic peptide antibiotic, that was used before in the second line treatment of multidrugresistant tuberculosis. The known molecular mecanism of action is the inhibition of the elongation phase of protein synthesis. In this study, we used the microorganism E. coli, because it is a gold standard to study antibiotic mechanism of action and a spectrophotometer, called stopped-flow, to show conformational changes of the initiation factor 3 (IF3). It was shown that binding of capreomycin to the small subunit 30S brings IF3 domains into proximity, which seems like a decrease of fluorescence. This effect was found to be concentration dependent, finding a greater fluorescence decrease when the concentration of capreomycin was higher. When initiation factor 1 (IF1) was added to the 30S – IF3DL complex with capreomycin, a further decrease of inter domain distance was observed; yet, slightly less than in absence of IF1. The kinetic analysis of capreomycin binding in the presence or absence of IF1 shows that IF3DL inter domain distance reduction is IF1 – independent. In conclusion, capreomycin induces a reduction of the inter domain distance of IF3, which is expected to perturb its main fidelity function. / Tesis