Tuberculosis is the world's largest cause of death from an infectious agent. Treatment is by an extended period of combination chemotherapy. Drug resistance is an increasing problem in tuberculosis therapy, particularly to the frontline anti-tubercular drug isoniazid (INH). Recombinant arylamine N-acetyltransferase (NAT) of Mycobacterium tuberculosis N-acetylates INH using the cofactor Acetyl Coenzyme A. NAT from M. tuberculosis is a polymorphic enzyme and also acetylates INH in vivo. Acetylated INH is inactive therapeutically against M. tuberculosis both in vivo and in vitro. The acetylation of isoniazid in the mycobacterial cell may compete with the activation of INH by the catalase-peroxidase, katG, and hence contribute to INH resistance in clinical isolates. Inhibition of NAT in M. tuberculosis may thus increase the efficacy of INH therapy. A novel assay based around the detection of free Coenzyme A released during the acetylation reaction was used to determine the substrate specificity of recombinant NAT from the related Mycobacterium M. smegmatis (MSNAT). A relationship was observed between the lipophilicity of simple arylamine substrates and the rate of acetylation by MSNAT. Several MSNAT substrates possess antibacterial activity. The assay could also be used to screen compound libraries for MSNAT inhibitors. Synthesis of seventeen thiazolidinedione sultams in collaboration with Dr.Vickers (Dyson Perrins), identified as weak inhibitors of MSNAT, gave a minimum competitive inhibitory constant of 14μM. Screening a library of 5,074 drug-like compounds for inhibition of MSNAT identified thirteen compounds with semi-maximal inhibition constants (IC₅₀) of below 10μM. Based on this, fifteen maleimides were synthesised and were irreversible inhibitors of MSNAT with submicromolar potency. Similarly, ninety-six aminothiazoles were synthesised by Dr. Vickers and were uncompetitive inhibitors of MSNAT with a minimum IC₅₀ of 1.5μM. The most potent aminothiazole showed no effect on the growth of M. smegmatis or M. bovis BCG or the sensitivity of the bacteria to isoniazid. However the aminothiazoles were shown not to penetrate the cells.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M. Sc.)(Biochemistry)--University of Pretoria, 2006. / Includes bibliographical references. Available on the Internet via the World Wide Web.

Dissertation ( PhD)--University of Stellenbsoch, 2007. / Bibliography. Also available via the Internet.

Thesis (M.S.)--Michigan State University. Dept. of Large Animal Clinical Sciences, 2008. / Title from PDF t.p. (viewed on July 29, 2009) Includes bibliographical references. Also issued in print.

Zugl.: Oldenburg, Universiẗat, Diss., 2009.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Zugl.: Giessen, Univ., Diss., 2008

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-29Bitstream added on 2014-06-13T19:22:40Z : No. of bitstreams: 1 miyata_m_dr_arafcf.pdf: 1125608 bytes, checksum: 1f8f6a7c62d2f6a383319def72bb3a51 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / De acordo com a Organização Mundial de Saúde em 2009, 9,27 milhões de novos casos de tuberculose ocorreram em 2007. Destes novos casos, 4,9% eram multidroga resistentes. Muitas pesquisas são realizadas na procura de novas drogas com atividade contra o bacilo da tuberculose, havendo então a necessidade de se entender os mecanismos de ação destes novos compostos. Este projeto objetivou propiciar ferramentas para compreender um pouco mais sobre os mecanismos de ação de novas drogas. Isolados clínicos de M. tuberculosis foram caracterizados quanto ao seu perfil de susceptibilidade aos fármacos do esquema terapêutico e foram determinadas as mutações responsáveis por estas resistências. Com os isolados caracterizados, foi construído um painel de M. tuberculosis. Pelo REMA, os isolados foram analisados quanto ao seu perfil de susceptibilidade aos fármacos (INH, RMP, STR e ETB) e avaliados quanto à presença de mutações nos genes de resistência (inhA, katG, ahpC, rpoβ, rpsL, rrs e embB) empregando a PCR-SSCP. Pelo REMA foram avaliados 80 isolados clínicos, sendo observada a resistência a INH em 74,7%, a RMP em 51,2%, a STR em 53,7% e ao ETB em 58,7%. Nos isolados resistentes, a porcentagem de mutações encontradas nos genes foi de 20,6% para inhA, 50% para katG, 6,3% para ahpC, 60% para rpoβ, 20% para rpsL e 0% para rrs e embB. Um painel com 12 isolados foi testado frente a três novos compostos, dois derivados de INH (Cu-INH1 e Cu-INH2) e um de RMP (Cu-RMP). Verificou-se que os isolados resistentes a INH foram também resistentes a Cu-INH1 e Cu-INH2. A mesma situação foi verificada em relação à RMP, com o composto Cu-RMP. Provavelmente, estes novos compostos têm os mesmos mecanismos de ação da INH e da RMP, que são os fármacos que lhes deram origem / According to World Health Organization in 2009, 9.27 million new TB cases occurred in 2007. Among these new cases, 4.9% were multidrug resistant. Many surveys are conducted in the search for new drugs with activity against the tuberculosis bacillus, therefore there is a need to understand the action mechanism of these new compounds. This project aimed to provide tools to understand about the action mechanisms of new drugs. M. tuberculosis clinical isolates were analyzed for their susceptibility profile to drugs, mutations responsible for resistance and a panel of these characterized isolates. The isolates were analyzed for susceptibility profile to drugs (INH, RIF, STR and ETB) and evaluated for presence of mutations in the resistance genes (inhA, katG, ahpC, rpoβ, rpsL, rrs and embB) applying the PCR-SSCP. REMA evaluated 85 clinical isolates and the resistance was observed in 74.7% to INH, 51.5% to RIF, 53.7% to STR and 58.7% to ETB. In the resistant isolates, percentage of mutations found in the genes was 20.6% for inhA, 50% for katG, 6.3% for ahpC, 60% for rpoβ, 20% for rpsL and 0% for rrs and embB. A panel of 12 isolates was tested against three new compounds, two INH-derivatives (Cu-INH1 and Cu-INH2) and one RMP-derivative (Cu-RMP). The isolates resistant to INH were also resistant to Cu-INH1 and Cu-INH2 compounds. The same situation was verified in relation to the RMP with the Cu-RMP compound, indicating that probably these three new compounds have the same action mechanism of INH and RMP drugs

Considerando as limitações do diagnóstico convencional da tuberculose (TB) e o avanço das tecnologias de diagnóstico, nosso grupo desenvolveu o ensaio in house TaqMan-IS6110 para a detecção do DNA do complexo Mycobacterium tuberculosis. O objetivo deste estudo foi determinar a acurácia do Detect-TB e do ensaio TaqMan-IS6110 em comparação com os métodos de diagnóstico padrão ouro para a TB e o desfecho clínico. Um total de 216 amostras clínicas de escarro espontâneo de pacientes com suspeita de TB foram incluídas. A sensibilidade (SE) e especificidade (SP) do Detect-TB em comparação com a baciloscopia e/ou cultura (n= 109) foram de 89,4% e 70,4%. A SE e SP do Detect-TB em comparação com o desfecho clínico (n= 197) foram de 86,6% e 66,4%. A SE e SP do ensaio TaqMan-IS6110 em comparação com a baciloscopia e/ou cultura (n= 109) foram 92,1% e 62,0%. A SE e SP do ensaio TaqMan-IS6110 em comparação com o desfecho clínico (n= 197) foram de 93,3% e 55,5%. A concordância entre os testes foi demonstrada pelo índice Kappa de 0,56 (P < 0,001) (n= 197). O Detect-TB e o ensaio TaqMan-IS6110 apresentaram valores de SE consistentes e SP inferiores quando comparados com outros testes moleculares in house e kits comerciais. O ensaio TaqMan-IS6110 deve ser avaliado com um maior número amostral para poder ser validado e para, eventualmente, poder ser implementado comercialmente. / Considering the limitations of conventional tuberculosis (TB) diagnosis and the advancement of diagnostic technologies, our group developed an in house IS6110-TaqMan assay for the detection of Mycobacterium tuberculosis complex DNA. The aim of the study was to determine the accuracy of the commercial molecular diagnostic assay Detect-TB and the IS6110-TaqMan assay compared to the TB gold standard diagnostic methods and the clinical outcome. A total of 216 clinical specimens of spontaneous sputum from TB suspected patients were included. Detect-TB sensitivity (SE) and specificity (SP) compared with the smear microscopy and/or culture (n= 109) were 89.4% and 70.4%. Detect-TB SE and SP compared with the clinical outcome (n= 197) were 86.6% and 66.4%. IS6110-TaqMan assay SE and SP compared with the smear microscopy and/or culture (n= 109) were 92.1% and 62.0%. IS6110-TaqMan assay SE and SP compared with the clinical outcome (n= 197) were 93.3% and 55.5%. The concordance among the tests were demonstrated by the Kappa index of 0.56 (P < 0.001) (n= 197). IS6110-TaqMan assay and Detect-TB showed consistent SE and lower SP when compared with other current molecular in house and commercial kits. IS6110-TaqMan assay should be evaluated with a larger number of samples to be validated and may eventually be implemented commercially.