Includes bibliographical references / New chemotherapeutics are urgently needed to combat Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). The development of compounds that could potentiate the activity of known antimycobacterial drugs is a relatively unexplored approach to new TB drug discovery. This study aimed to generate metabolites of chlorpromazine (CPZ), a phenothiazine with demonstrated in vitro activity against Mtb, and to investigate their potential utility in combination with anti-TB drugs. 7-HydroxyCPZ (M2), CPZ-N-oxide (M3), CPZ sulfoxide (M1), nor-CPZ (M5), nor-CPZ sulfoxide (M6b) and CPZ-N-S-dioxide (M4b) were generated from CPZ using various biotransformation systems and identified by Liquid Chromatography - Mass Spectrometry (LC/MS). The identity of M2 was confirmed with reference to a 7-hydroxyCPZ standard. M3, M1, M5, M6b and M4b were synthesized de novo and used to identify the metabolites generated in the biotransformation samples. Individually, CPZ and its metabolites (M2, M3, M5) were weakly active (MIC99 >50μM) against M. smegmatis (Msm) and Mtb while M1, M6b & M4b did not exhibit a MIC99 even at very high concentrations. Generally, an improvement in activity was observed where CPZ or its metabolites were used in combination with known anti-TB drugs. The combinations that exhibited a fractional inhibition concentration index (FICI) of < 0.5 were defined as synergistic. A combination of M2 and spectinomycin (SPEC) exhibited the highest synergism against Msm (FICI 0.19) and Mtb (FICI 0.13). In vitro assays established that CPZ and M2 are bactericidal against Mtb whereas M3 and M5 are bacteriostatic on their own. In combination assays, the use of RIF with M3 and M5, bedaquiline (BDQ) with M2, and SPEC with M3 were bactericidal. At 140μM, CPZ and M1, M2, M3 treated samples exhibited a 2-fold up-regulation of the cydA (Rv1623c) gene which encodes an essential subunit of the cytochrome bd-type menaquinol oxidase in Mtb. The same observation was made for RIF/M2 and RIF/M5 treated samples. These results suggest that the metabolites retain the mechanism of action (MoA) as the parental CPZ. The Mtb 16S rRNA gene, rrs (MTB000019) was identified as the biological target for SPEC. This brought into perspective the underlying mechanisms at play when SPEC is used in combination with CPZ, its metabolites or other drugs, against mycobacteria. This study establishes the utility of combination assays in confirming the active metabolite(s) of known drugs and provides proof of concept data to support follow-up investigations of CPZ and its metabolites as potential compounds for novel combination therapies for anti-TB drug development.

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Includes bibliographical references / Tuberculosis (TB) is one of the major infectious diseases and epidemics in the world. It is responsible for severe morbidity and mortality rates, especially in poor and resource-deficient countries. According to the World Health Organization 2014 report, about one third of the world's population is infected with tuberculosis and about 10-15% is co-infected with HIV, which further complicates the TB epidemic. Tuberculosis claims 2-3 million lives every year and is one of the biggest social and financial burdens on many countries. The disease is treatable but has been hampered by the emergence of drug resistance in the causative bacterium, Mycobacterium tuberculosis (Mtb). Resistant strains of Mtb counter the efficacy of various anti-TB drugs via mechanisms that help it overcome the toxic and inhibitory effects of these drugs. These mechanisms include mutation, enzymatic drug degradation, target modification and drug efflux. Drug efflux by efflux pumps (EPs) is one of the major mechanisms responsible for the development of drug tolerance leading to the emergence of drug resistance. These efflux pumps are regulated by the house keeping proteins present in the cell membrane of Mtb and perform a pre-existing role of rescuing the Mtb from toxic agents. These EPs extrude structurally unrelated compounds from the cell including anti-TB drugs and reduce the drug concentration to sub-inhibitory levels and aid Mtb in developing resistance. Therefore, development of antimycobacterials that target EPs and reduce their activity can be a viable strategy to reduce the global TB burden and counter the emergence of resistance. Many strategies have been used to counter the EP-mediated resistance in Mtb. In this study, two strategies were employed: (i) the development of efflux pump inhibitors (EPIs) via structural modification of a known efflux pump inhibitor, verapamil (VER), and the development of hybrid efflux pump inhibitors (HEPIs) incorporating a VER motif; and (ii) the development of antimycobacterial agents based on covalent linking or attachment of efflux pump inhibitor moieties to an anti-TB drug. These agents are termed reversed anti-TB agents and are based on isoniazid for this study.

Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Bc. Lenka Králíková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma: Atypical mycobacteria as causative agents of human diseases The aim of this diploma thesis is to summarize the history of mycobacteria, their classification, epidemiology of nontuberculous infections, clinical significance, diagnosis, resistance, and treatment options. Within the epidemiology, the way of managing tuberculosis and non-tuberculous mycobacterioses is compared. Mycobacterioses are rare diseases, which incidence is gradually increasing with the increasing number of immunocompromised patients. Based on the spot of infection, we divide them into lymphadenitis, skin lesions, lung disease and disseminated infections. The most frequently isolated pathogens are Mycobacterium avium complex, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium marinum and Mycobacterium kansasii. The experimental part is devoted to the antimycobacterial activity of newly synthesized compounds, which could find application in the future in the treatment of tuberculosis and other mycobacterioses. Substance testing was performed on strains of the genus Mycobacterium...