Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis. The present study initially focused on the characterization of a family of membrane anchored, cell wall associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high levels of gamma interferon secretion by T-cells in response to these proteases were observed in Mantoux positive individuals. The absence of any detectable protease activity lead to a protein sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein processing proteases. To identify possible substrates for these proteases, the genome sequence regions surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part of a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M. tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigen family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In silico analysis of finished and unfinished genome sequencing data revealed the presence of orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have established the duplication order of the gene clusters and demonstrated that gene cluster region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could be found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple duplications of this cluster have occurred and have been maintained only within the genomes of members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene family (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins (secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putative ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the observation that members of the ESAT-6 family are secreted without the normal sec-dependent secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins function together to form a transport system for the secretion of the members of the ESAT-6 protein family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was also identified and its activity characterized. Subsequent secretion analyses results have shown that secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a number of features of quorum sensing and lantibiotic operons, and an extensive review of the literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as signaling molecules and may be involved in the regulation of expression of genes during intracellular residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE gene families (members of which is found situated in the ESAT-6 gene clusters) were investigated. This investigation revealed that the expansion of these families are linked to the duplications of the ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences distinguishing these two groups of mycobacteria. In conclusion, this study has highlighted numerous important aspects of mycobacterial genomics and has greatly contributed to the current body of knowledge concerning the role of proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Children contribute little to disease transmission and the maintenance of the tuberculosis epidemic, but they constitute a significant proportion of the total tuberculosis (TB) caseload and experience considerable morbidity and mortality in endemic areas, despite the availability of cheap and effective treatment. The difficulty of diagnosing childhood tuberculosis is one of the major obstacles that hinder the provision of antituberculosis treatment to children in endemic areas. The diagnosis of childhood tuberculosis is complicated by the lack of a practical gold standard, as bacteriologic specimens are difficult to collect and the yield is low. In non-endemic countries the diagnosis of childhood tuberculosis is based on the triad of: 1) exposure to an adult index case, 2) a positive tuberculin skin test, and 3) suggestive radiographic signs. However, the triad has limited value in endemic areas where exposure to and/or infection with Mycobacterium tuberculosis is common, and chest radiography is rarely available. The objective of this dissertation was to address the diagnostic dilemma faced by health professionals in endemic areas with limited resources, where children currently have poor access to chemoprophylaxis and antituberculosis treatment. We first clarified basic disease concepts, through a critical review of the pre-chemotherapy literature that documented the natural history of childhood tuberculosis. Three central concepts were identified; 1) the importance of accurate case definition, 2) the relevance of risk stratification, and 3) the diverse spectrum of disease, which necessitates accurate disease classification. The importance of accurate case definition is illustrated by the fact that isolated hilar adenopathy, considered the principal radiographic sign of primary tuberculosis, becomes transiently visible in the majority of children following recent primary infection. Our analysis of the natural history of childhood tuberculosis allowed accurate quantification of the risk to progress to disease following primary infection with M.tuberculosis. This demonstrated that the risk depends mainly on the age and/or immune-status of the child, the time since primary infection occurred and the presence or absence of symptoms. After analysing these historic studies, we proceeded to document the burden of childhood tuberculosis in an endemic area. We first conducted a retrospective study to describe current diagnostic practices and demonstrated almost exclusive reliance on chest radiography. We then calculated the burden of childhood tuberculosis in a prospective descriptive study. The corrected tuberculosis incidence rate in children was 407/100 000/year and children with severe forms of disease, such as disseminated (miliary) tuberculosis and/or tuberculous meningitis, were rarely recorded in the TB treatment register used for routine community-based surveillance. An additional obstacle to progress in the field of childhood tuberculosis has been the lack of standard descriptive terminology. Following a careful review of the literature, we proposed a radiological classification of childhood intra-thoracic tuberculosis and explored the different pathologic mechanisms that underlie these diverse disease manifestations. We then conducted a prospective descriptive study to document the disease spectrum in children treated for tuberculosis in an endemic area. The disease patterns observed were consistent with those described in the pre-chemotherapy literature. In addition, we demonstrated that bacteriologic confirmation may be achieved in the majority of children with intra-thoracic tuberculosis, in highly endemic settings. Finally we developed a novel symptom-based approach to diagnose pulmonary tuberculosis in children from endemic areas with limited resources. We followed a step-wise approach by first conducting a community-based survey to document the prevalence of symptoms traditionally associated with tuberculosis in a random selection of children from an endemic area. The survey demonstrated that poorly defined symptoms offer poor diagnostic value. The second step was to evaluate the diagnostic value of well-defined (persistent, non-remitting) symptoms in a small prospective study. Well-defined symptoms demonstrated good diagnostic value, but these promising results required further validation. As a final step, we validated the diagnostic value of a novel symptom-based approach in a large prospective, community-based study. In this study, a simple symptom-based approach diagnosed childhood pulmonary tuberculosis with a remarkable degree of accuracy, particularly in HIV-uninfected children older than 3 years of age. This novel diagnostic approach offers the exciting prospect of extending antituberculosis treatment to children in endemic areas with limited resources, where current treatment access is poor. / AFRIKAANSE OPSOMMING: Tuberkulose beheer programme plaas feitlik geen klem op die behandeling van kinders nie, omdat kindertuberkulose selde aansteeklik is en die persepsie bestaan dat kinders slegs in raar gevalle ernstig siek word. Tuberkulose lewer egter ‘n betekenisvole bydrae tot kindermorbiditeit en mortaliteit in endemiese areas, terwyl dit ‘n maklik behandelbare siekte is. Kindertuberkulose is moeilik om te diagnoseer en dit is ‘n belangrike faktor wat daartoe bydra dat kinders dikwels nie antituberkulose behandeling ontvang wanneer hulle dit benodig nie. Die diagnose van kindertuberkulose is moeilik, omdat die organisme selde aangetoon kan word. In nie endemiese areas word kindertuberkulose dikwels gediagnoseer na aanleiding van: 1) blootstelling aan ‘n volwasse indeks geval, 2) ‘n positiewe tuberkulien veltoets, en 3) die teenwoordigheid van radiologiese tekens suggestief van tuberkulose. Hierdie benadering het defnitiewe tekortkominge in endemiese areas, waar blootstelling aan en infeksie met Mycobacterium tuberculosis algemeen is. Gevolglik berus die diagnose van kindertuberkulose hoofsaaklik op die subjektiewe interpretasie van die borskasplaat, wat welbekende tekortkominge het en verder is radiologiese toetse dikwels nie beskikbaar in hierdie areas nie. Die doel van die navorsingsprojek was om die dilemma rondom die diagnose van kindertuberkulose in endemiese areas aan te spreek. Eerstens is basiese siektekonsepte uitsorteer deur ‘n kritiese oorsig van studies uit die pre-chemoterapie era. Hierdie kosbare studies het die natuurlike verloop van tuberkulose in kinders beskryf, nog voordat antituberkulose middels beskikbaar was. Drie sentrale konsepte is geidentifiseer; 1) die belang van akkurate siekte definisie, 2) die relevansie van risiko stratifikasie en 3) die diverse spektrum van patologie wat akkurate siekte klassifikasie noodsaak. Die belang van akkurate siekte definisie word geïllustreer deur die feit dat geïsoleerde hilêre adenopatie ‘n verbygaande verskynsel is in die meerderheid van kinders kort na primêre infeksie. Ons analise het daarop gefokus om die risiko om siekte te ontwikkel nadat primêre infeksie met M.tuberculosis plaasgevind het, te kwantifiseer. Die hoof risiko faktore was; 1) die ouderdom en/of immuunstatus van die kind, 2) die tydsverloop sedert infeksie, en 3) die teenwoordigheid van simptome al dan nie. Hierna het ons die siektelas wat tuberkulose vandag op kinders in endemiese areas plaas gedokumenteer. Ons het eers die huidige diagnostiese praktyke geëvaluaeer in ‘n retrospektiewe studie en toe ‘n prospektiewe beskrywende studie gedoen om die siektelas so akkuraat as moontlik te meet. Die insidensie van kindertuberkulose was hoog (>400/100 000/jaar), selfs na korreksie vir kinders wat ontoepaslik behandeling ontvang het. Verder is gevind dat die meerderheid van kinders met ernstige siekte toestande soos miliêre tuberkulose en/of meningitis, nie in roetine moniterings data reflekteer word nie. ‘n Bykomende struikelblok in kindertuberkulose is die gebrek aan standaard beskrywende terminologie. Om dit te bevorder ontwikkel ons ‘n nuwe radiologiese klassifikasie van intra-torakale kindertuberkulose en beskryf ons die verskillende patologiese meganismes onderliggend tot hierdie uiteenlopende siektebeelde. Daarna dokumenteer ons die volledige spektum van kindertuberkulose in ‘n endemiese area en demonstreer dat die siektepatrone wat ons vandag observeer soortgelyk is aan die wat in die pre-chemoterapie literatuur beskryf is. Ons toon ook dat bakteriologiese bevestiging moontlik blyk te wees in die meerderheid van kinders wat vir intra-torakale tuberkulose behandel word in endemiese areas. Nadat ons duidelikheid verkry het oor die basiese siektekonsepte, siekte klassifikasie en die siektelading in ons omgewing, kon ons op die ontwikkeling van ‘n simptoom-gebaseerde benadering tot die diagnose van kindertuberkulose fokus. Ons het ‘n stapsgewyse benadering gevolg. Die eerste stap was om die voorkoms van simptome wat gebruiklik met tuberkulose vereenselwig word te dokumenteer in ‘n ewekansige groep kinders. Die gemeenskapsopname het getoon dat swak gedefiniëerde simptome swak diagnostiese waarde bied. Die tweede stap was om vas te stel of verbeterde simptoom definisie die diagnostiese waarde kan verbeter. ‘n Klein prospektiewe studie het getoon dat goed gedefiniëerde simptome (persisterende simptome van onlangse aankoms) goeie diagnostiese waarde bied. Die finale stap was om hierdie belowende benadering formeel te toets in ‘n groot prospektiewe, gemeenskapsgebaseerde studie. Hierdie studie het getoon dat ‘n eenvoudige simptoom-gebaseerde benadering pulmonale tuberkulose met goeie akkuraatheid kan diagnoseer, veral in HIV-ongeïnfekteerde kinders wat ouer is as 3 jaar. Hierdie nuwe diagnostiese benadering bied die moontlikheid om antituberkulose behandeling te voorsien aan kinders in endemiese areas wat tans feitlik geen behandeling ontvang nie.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

The pathogenic bacterium Mycobacterium tuberculosis (Mt) is the causative agent of tuberculosis, a widespread and fatal infectious disease. Today, treatment against tuberculosis involves a combination of drugs, which need to be taken for at least six months and which often causes severe side effects. Therefore, new drugs that are more effective and that give fewer side effects are needed. A characteristic feature of the Mt bacterium is its very complex and thick cell wall, which prevents many potential drug molecules from penetrating it. Inhibiting any one of the enzymes that are involved in its biosynthesis would therefore seem to be a good strategy for eliminating the Mt bacteria. The aim of this study was to characterize four enzymes involved in Mt cell wall biosynthesis. In order to do that, they were produced recombinantly in E. coli and purified. Crystallization experiments were set up in order to produce diffracting crystals, with the aim of structure determination and drug design.

Mycobacterium tuberculosis (Mtb), a pathogenic bacterium, is the causative agent in the vast majority of human tuberculosis (TB) cases. Nearly one-third of the world’s population has been affected by TB and annually two million deaths result from the disease. Because of the high cost of medication for a long term treatment with multiple drugs and the increase of multidrug-resistant Mtb strains, faster-acting drugs and more effective vaccines are urgently demanded. Several metabolic pathways of Mtb are attractive for identifying novel drug targets against TB. Hence, a high quality genome-scale metabolic network of Mtb (HQMtb) was reconstructed to investigate its whole metabolism and explore for new drug targets. The HQMtb metabolic network was constructed using an unbiased approach by extracting gene annotation information from various databases and consolidating the data with information from literature. The HQMtb consists of 686 genes, 607 intracellular reactions, 734 metabolites and 471 E.C. numbers, 27 of which are incomplete. The HQMtb was compared with two recently published Mtb metabolic models, GSMN-TB by Beste et al. and iNJ661 model by Jamshidi and Palsson. Due to the different reconstruction methods used, the three models have different characteristics. The 68 new genes and 80 new E.C. numbers were found only in the HQMtb and resulting in approximately 52 new metabolic reactions located in various metabolic pathways, for example biosynthesis of steroid, fatty acid metabolism, and TCA cycle. Through a comparison of HQMtb with a previously published human metabolic network (EHMN) in terms of protein signatures, 42 Mtb metabolic genes were proposed as new drug targets based on two criteria: (a) their protein functional sites do not match with any human protein functional sites; (b) they are essential genes. Interestingly, 13 of them are found in a list of current validated drug targets. Among all proposed drug targets, Rv0189c, Rv3001c and Rv3607c are of interest to be tested in the laboratory because they were also proposed as drug target candidates from two research groups using different methods.

Les phospholipases et en particulier les phospholipases C sont d'importants facteurs de virulence chez de nombreuses bactéries pathogènes (C. perfringens, B. Cereus et P. aeruginosa). Cependant, peu de choses sont connues sur l'implication de ces enzymes dans le processus de virulence des mycobactéries. Bien que l'étude des mutants des phospholipases C de M. tuberculosis dans un modèle d'infection chez la souris ait permis de proposer une implication de ces protéines dans la virulence de ce bacille, leurs propriétés biochimiques, leur mode d'action et leur rôle physiologique exact restent à élucider. Ce manque de données biochimiques sur les phospholipases mycobactériennes peuvent être attribuée à la difficulté à produire et à purifier des quantités importantes de ces enzymes. Dans le but de mieux caractériser le rôle physiologique des phospholipase mycobactériennes, l'objectif de ma thèse a été de mettre au point des conditions d'expression hétérologue permettant la production des phospholipases C mycobactériennes recombinantes (rPLC) dans différents systèmes d'expression (E. coli, Pichia pastoris et baculovirus/cellules d'insectes). Ces systèmes d'expression n'ayant pas donné des résultats satisfaisants, nous avons développé une méthode efficace d'expression de ces protéines en utilisant M. smegmatis.Ce système d'expression nous a permis de produire et de purifier les quate PLC (PLC-A, PLC-B, PLC-C et PLC-D) de M. tuberculosis et la PLC de M. Abscessus sous forme soluble et active. Nous avons pour la première fois montré que ces protéines purifiées avaient un effet cytotoxique sur les macrophages de souris en culture mais ne présentaient aucune activité hémolytique. en utilisant des marquages radioactifs, nous avons confirmé que l'effet cytotoxique observé était lité à l'hydrolyse des phospholipides des membranaires des cellules hôtes. Pour la première fois, nous avons pu confirmer que ces PLC sont directement impliquées dans le processus d'infection et de virulence.Un autre aspect de mon travail de thèse a concerné l'étude de deux autres protéines sécrétées par M. tuberculosis appartenant à la famille des cutinases : la Rv1984c et la Rv3452. Après les avoir produites et purifiées chez E. Coli, nouq avons montré que malgré ces deux protéines présentent 50% d'identité de séquence en acides aminés, elles ont des spécificités de substrat différentes et probablement un rôle physiologique différent. La Rv1984c est une lipase capacle d'hydrolyser des lipides à chaines moyennes, alors que la Rv3452 est une phospholipase de type A2 et est capable d'induire la lyse de macrophage de souris en culture. / Phospholipases, particularly phospholipases C, are important virulence factors in several pathogenic bacteria (C. perfringens, B. cereus, L. monocytogenese and P. aeruginosa). However, little is know on the involvement of thses enzymes in mycobacteria pathogenesis. Although study on M. tuberculosis phospholipases C mutants in a mouse aerosol model of infection gave rise to the contribution of these proteins in virulence process, but their exact biochemical properties, mechanism of action and physiological role remain to be elucidated. This lack of data on mycobacterial phospholipases is mainly due to the difficulty to produce and purify these enzymes in large scale.With the aim to better characterise the physiological role of mycobacterial phospholipases, the main challenge of my thesis was to develop an efficient method for expression and purification of recombinant mycobacterial phospholipases C. Since no satisfactory results have been obtained with standard expression systems (E. coli, Pichia pastoris and baculovirus / insect cells), we develop a robust expression technique for these proteins using M. smegmatis as expression system.This allowed us to produce and purify all four PLC (PLC-A, PLC-B, PLC-C and PLC-D) of M. tuberculosis and the PLC of M. abscessus in soluble and active form. For the first time, we have show, that purified proteins have cytotoxic effect on mouse macrophages but have not haemolytic activity. Using radiolabelled lipids, we have confirmed that this first direct evidence that PLC are involved in infection and virulence processes. Another aspect of my thesis work concerned the study of two other secreted proteins of M. tuberculosis belonging to the cutinase family : the Rv 1984c ant the Rv3452. Recombinant proteins obtains in E. coli were found to have distinct substrate specificities and most likely distict physiological role, despite showing 50% amino acids sequence identity. Rv1984c is a lipase and is able to hydrolyse lipids with medium chains lengthn whereas Rv3452 is type A2, phospholipase and i able to induce macrophage lysis.

Describe la epidemiologia molecular de las cepas circulantes de Mycobacterium tuberculosis mediante PCR en tiempo real y análisis de alta resolución en especímenes clínicos del Hospital Nacional Arzobispo Loayza.

Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNy-dependent autophagy, respectively. Indeed, sustained expression and activation of PKR reduced the intracellular survival of Mtb, an effect that could be enhanced by IFNy treatment. We further demonstrate that the enhanced anti-mycobacterial activity of THP-PKR macrophages is mediated by a mechanism dependent on selective autophagy as indicated by increased levels of LC3-II that colocalize with intracellular Mtb. Consistent with this mechanism, inhibition of autophagolysosome maturation with bafilomycin A1 abrogated the ability of THP-PKR macrophages to limit replication of Mtb, whereas pharmacological activation of autophagy enhanced the anti-mycobacterial effect of PKR overexpression. As such, PKR represents a novel and attractive host target for development of HDT for TB, and our data suggest value in the design of more specific and potent activators of PKR.

Master of Science - Molecular Medicine and Haematology / The completion of the genome sequence of Mycobacterium tuberculosis strain H37Rv revealed that 10% of the coding capacity is devoted to two, large multigene families that are characterised by repeat sequences. These are the PE and PPE families that code for acidic, glycine rich proteins. A subgroup of the PE family is the polymorphic GC rich sequence (PGRS) gene subfamily. Genome comparisons of clinical isolates of M. tuberculosis have confirmed the polymorphic character of some of these genes suggesting they may be analogous to the contingency loci found in other pathogenic bacteria. Certain PE-PGRS proteins play a direct role in virulence in M. marinum, other PE-PGRS genes are cell surface associated, and some PE-PGRS proteins are variable surface antigens, supporting a potential role in host pathogen interactions. A reporter assay designed to investigate mutations in a PE-PGRS repeat-containing sequence was used to assess mutation rates in various M. smegmatis host strains by fluctuation analysis. A wide spectrum of mutations was observed and the evidence suggests that slipped-strand mispairing between proximal and distal PGRS sequences located in cis is the predominant type of mutational event at such loci. Moreover, slipped-strand mispairing at such loci occurs at a moderately higher rate than base substitution mutagenesis and is mediated by the normal replicative polymerase.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-01-16T11:14:58Z No. of bitstreams: 1 FERNANDA_SOUZA_MACCHI_DIS.pdf: 4481387 bytes, checksum: 59f26900156a5b5c1a267893bdc6f655 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-01-26T12:32:00Z (GMT) No. of bitstreams: 1 FERNANDA_SOUZA_MACCHI_DIS.pdf: 4481387 bytes, checksum: 59f26900156a5b5c1a267893bdc6f655 (MD5) / Made available in DSpace on 2018-01-26T12:40:54Z (GMT). No. of bitstreams: 1 FERNANDA_SOUZA_MACCHI_DIS.pdf: 4481387 bytes, checksum: 59f26900156a5b5c1a267893bdc6f655 (MD5) Previous issue date: 2017-11-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Using the classical hybridization approach series of 1H-benzo[d]imidazoles and 3,4-dihydroquinazolin-4-ones were synthesized and evaluated as inhibitors of Mycobacterium tuberculosis growth. Chemical modifications and structure-activity relationship studies yielding potent antitubercular agents with minimum inhibitory concentration values in submicromolar range. Further, the synthesized compounds were active against drug-resistant strains and were devoid of apparent toxicity to HepG2, HaCat, and Vero cells. In addition, some 3,4-dihydroquinazolin-4-ones showed low risk of cardiac toxicity, no signals of neurotoxicity or morphological alteration in zebrafish (Danio rerio) models. Therefore, these data denote that this class of molecules may furnish candidates for future development of novel anti-TB drug alternatives. / Usando a abordagem cl?ssica de hibrida??o molecular, s?ries de 1H-benzo[d]imidaz?is e 3,4-diidroquinazolin-4-onas foram sintetizadas e ensaiadas como inibidores de crescimento de Mycobacterium tuberculosis. Modifica??es qu?micas e estudos de rela??o estrutura-atividade nos conduziram a potentes agentes antituberculose com valores submicromolares de concentra??o inibit?ria m?nima. Os compostos sintetizados tamb?m foram ativos contra cepas resistentes ? f?rmacos e demonstraram desprovida citotoxicidade aparente em c?lulas HepG2, HaCat e Vero. Al?m disso, algumas 3,4-diidroquinazolin-4-onas apresentaram baixo risco de toxicidade card?aca, e nenhum sinal de neurotoxicidade ou altera??o morfol?gica em modelo de peixe-zebra (Danio rerio). Sendo assim, os resultados indicam que essa classe de mol?cular pode fornecer condidatos para o desenvolvimento futuro de novos f?rmacos contra a tuberculose.