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Type: Theses
Title: Novel inhibitors for biotin biosynthesis pathway in Mycobacterium tuberculosis
Author: Salaemae, Wanisa
Issue Date: 2015
School/Discipline: School of Molecular and Biomedical Science
Abstract: Tuberculosis (TB) ranks alongside HIV-AIDS and malaria as one of the leading causes of death by infectious disease worldwide. The 2015 Millennium Development Goals (MDGs) for reducing the mortality rate and the incidence of new patients to 50% of the 1990 incidences have nearly been reached. However, the prevalence of multidrug-resistant TB (MDR-TB) remains well off-track and needs to be addressed as a public health crisis. Within the past 40 years only one new anti-TB drug with a unique mode of action, namely bedaquiline (FDA approved in 2012), has become available for drug resistant strains of TB but even this has concerning side effects. Clearly, there is an urgent need for safer drugs that have no pre-existing resistance mechanism to terminate the global prevalence of drug resistance TB. Biotin biosynthesis has been proposed as a promising druggable target for anti-TB drug discovery. Biotin is an essential metabolite required for growth of all living cells. Biotin is synthesized de novo in microorganism, plants, and fungi. The absence of this metabolic pathway in humans makes biotin biosynthesis attractive for antibiotic discovery. In particular, biotin biosynthesis plays an important metabolic role as the sole source of biotin in all stages of the tuberculi life cycle due to the lack of biotin transporter. Biotin is intimately associated with lipid synthesis where the products form key components of the cell membrane that is critical for bacterial survival. Therefore, enzymes involving in lipid synthesis and biotin biosynthesis have been designated as an excellent target for the development of new anti-TB drugs to combat drug resistant TB. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of the biotin biosynthetic pathway. It was selected as a target for screening inhibitors for M. tuberculosis biotin biosynthesis in this study due to its essential role in the growth and virulence of tuberculi. X-ray crystal structures of M. tuberculosis DTBS (MtDTBS) reveals two preformed adjacent ligand-binding pockets that allowed DAPA and NTP substrates to bind independently, making both pockets attractive for drug discovery. Enabling technologies were developed for the characterization of DTBS enzymes, including in silico screening coupled with X-ray crystal structures and three new facile assays for identifying ligand binding in the NTP pocket, namely an enzyme assay, a competitive ATP-binding assay and surface plasmon resonance (SPR) analysis. Unexpectedly, MtDTBS was shown to have broad specificity for a variety of nucleotide triphosphates, although the enzyme had the highest affinity for CTP in competitive binding and SPR assays. For the first time, X-ray crystal structure of MtDTBS bound to a nucleotide triphosphate (CTP) has been reported, showing that the nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. These novel findings for MtDTBS are in contrast to other DTBS orthologs, for example Escherichia coli DTBS (EcDTBS) preferentially binds ATP primarily through hydrogen-bonds between the adenine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role of asparagine at position 175 in E. coli DTBS that excludes binding of other nucleotide triphosphates. Due to the absence of the gate-keeper at an equivalent position, MtDTBS thus has the broad specificity to multiple types of nucleotide triphosphates. The X-ray crystal structure of MtDTBS in complex with CTP was used in an in silico, fragment-based approach to drug discovery. Compounds identified by in silico docking were verified using an SPR binding assay and DTBS enzyme assay. Total six hits (namely CT6, CT7, B1, B3, B7, and B9) were identified that were predicted to bind to the protein “hot spot” located in the phosphate-binding loop at the junction of the two ligand binding pockets. Lineweaver-Burk analysis revealed one compound, gemcitabine, was competitive against DAPA and ATP. The low molecular weight (< 300 Da), low chemical complexity, and good ligand efficiency (LE) (0.2-0.3 kcal/mol/heavy atom) of the hits make them attractive targets for chemical development into more drug-like leads. Interestingly, the anti-cancer gemcitabine CT6 clearly showed in vitro inhibitory activity against MtDTBS, suggesting an application of this existing drug as a new anti-TB agent. As proof of concept, the potential optimization of leads has been proposed via merging CT6 with DAPA carbamate in order to avoid potential toxicity that might cause through off-target of human NTP-dependent enzymes. Finally, the potential transcriptional regulation of M. tuberculosis biotin biosynthesis has been firstly proposed in order to understand the biotin metabolism of tuberculi and to combat TB effectively.
Advisor: Booker, Grant William
Polyak, Steven William
Dissertation Note: Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Molecular and Biomedical Science, 2015.
Keywords: biotin biosynthesis
dethiobiotin synthetase
mycobacterium tuberculosis
fragment based drug discovery
Research by Publication
Provenance: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
DOI: 10.4225/55/590010387b74c
Appears in Collections:Research Theses

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