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  • Escaping Bio-Assay Guided Isolation: Nature's Tools for Chemical Biology

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    Elnaas_Ali Ramadan_Final Thesis_Redacted.pdf (14.08Mb)
    Author(s)
    Elnaas, Ali Ramadan
    Primary Supervisor
    Quinn, Ronald J
    Other Supervisors
    Grice, Irwin D
    Liu, Miaomiao
    Year published
    2021-08-11
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    Abstract
    World Health Organisation (WHO) announced in 2016 that TB is a top infectious disease killer worldwide with emerging multi drug resistance. Developing a new drug with different mechanisms is urgently needed. A successful outcome will significantly advance treatment of TB by identifying new and novel natural products that are biologically active against Mtb H37Rv and the recombinant mycobacterial lipoamide dehydrogenase (Lpd) enzyme. Identification of novel metabolites prior to commencing isolation will avoid re-isolation of known and inactive compounds and will save time and wasted effort in re-discovering the known. To ...
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    World Health Organisation (WHO) announced in 2016 that TB is a top infectious disease killer worldwide with emerging multi drug resistance. Developing a new drug with different mechanisms is urgently needed. A successful outcome will significantly advance treatment of TB by identifying new and novel natural products that are biologically active against Mtb H37Rv and the recombinant mycobacterial lipoamide dehydrogenase (Lpd) enzyme. Identification of novel metabolites prior to commencing isolation will avoid re-isolation of known and inactive compounds and will save time and wasted effort in re-discovering the known. To achieve this, a previous high-throughput screening assay was developed and tested 202,983 natural product fractions sourced from a diverse collection of marine invertebrates, plants and fungi. A set of 574 Fractions were active against Mtb H37Rv and LPD enzyme. In this work, 94 dried ground biota samples (47 plants and 47 marine) were collected from Australia, Papua New Guinea and China. Each sample was extracted through a Lead-Like Enhanced (LLE) Extraction protocol based on Lipinski’s Rule of 5 (Log P value ˂ 5) to produce drugs that can be taken orally. Each crude extract was fractionated through HPLC to give 5 fractions (470 fractions in total). The proton NMR at 800 MHz was obtained for all 470 fractions. The proton NMR spectra for the active fractions were analysed by PCA statistical analysis to investigate discrimination between the fractions. Taxonomy classification was carried on the 94 biota samples according to genus and species together with the HPLC profile and the proton NMR fingerprints of each sample. Six samples from different genera were chosen because of the different metabolites. Fraction 2 derived from plant Polyalthia sp. showed activity against Mtb H37Rv strain at 62.5 μge/μL. A known compound, altholactone, was identified from this fraction and showed activity towards M. tuberculosis strains mc26230, M. smegmatis mc2155, Mtb H37Ra and Mtb H37Rv at a minimum inhibitory concentration (MIC) of 64, 500, 26.9 and 137.9 μM respectively. Retrospective analysis of a target-based analysis against a TB proteome panel using native mass spectrometry, established that the active fraction was bound to the mycobacterial protein Rv1466. The binding affinity of the altholactone was confirmed with the Rv1466 protein with an estimated pseudo Kd of 42.0 ± 6.1 μM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vitro toxicity towards M. tuberculosis. Fraction 5 derived from plant Styrax faberi Perk. showed activity against Mtb at 1.25 μge/μL. Two new compounds demethoxy masutakeside I (1), demethoxy egonol glucoside (2) and five known compounds egonol gentiobioside, masutakeside I, egonol acetate, gonol-2-methylbutanoate and egonol (3-7, respectively) were isolated from anti TB active fraction. Demethoxy masutakeside I, demethoxy egonol glucoside, egonol gentiobioside and masutakeside I showed activity towards M. tuberculosis strain mc26230 with minimum inhibitory concentrations (MIC) of 256 μM, 256 μM, 128 μM and 128 μM, respectively. Retrospective analysis of a target-based assay against a TB proteome panel using native mass spectrometry, established that the active fraction was bound noncovalently to two mycobacterial proteins; phenylalanyl-tRNA synthetase alpha chain pheS and RNA polymerase sigma factor. Our findings established phenylalanyl-tRNA synthetase alpha chain pheS and RNA polymerase sigma factor as potential molecular targets of two new compounds (demethoxy masutakeside I and demethoxy egonol glucoside) and two known compounds (egonol gentiobioside and masutakeside I), which might be responsible for the low observed in vitro toxicity towards M. tuberculosis. Fraction 3 derived from marine species mauritiana, showed anti TB activity against Mtb H37Rv cell line with an MIC value of 2.5 μge/μL. The LLE fractionation protocol has led to isolation and identification of one major compound hymenidin (1). Fraction 4 derived from plant genus Flindersia, showed anti-TB activity against Mtb H37Rv with an MIC value of 2.5 μge/μL. The LLE fractionation protocol has led to isolation of one major compound γ-fagarine (2). Fraction 4 derived from the species novoguineensis teschn., showed anti Mtb activity with an MIC value of 10 μge/μL against Mtb H37Rv. The LLE fractionation led to isolation of one major compound goniothalamin (3). The compounds (1), (2) and (3) did not show activity against M. smegmatis even at high concentration. Compounds (1) and (2) showed very weak activity against Mtb mc26230 at MIC >512 μM, while lack of compound (3) prevented further testing. Fraction 4 derived from plant genus Euodia showed anti Mtb H37Rv activity with an MIC value of 5 μge/μL. Isolation and purification led to identification of five known alkaloids melicopidine (1), normelicopidine (2), melicopine (3), kokusaginine (6,7-dimethoxydictamine) (4) and evoxanthine (5). Structure of the isolated compounds were determined on the basis of intensive spectroscopic analyses of 1D , 2D NMR and MS technique.
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    Thesis Type
    Thesis (PhD Doctorate)
    Degree Program
    Doctor of Philosophy (PhD)
    School
    School of Environment and Sc
    DOI
    https://doi.org/10.25904/1912/4308
    Copyright Statement
    The author owns the copyright in this thesis, unless stated otherwise.
    Subject
    Mycobacterium tuberculosis
    Mtb
    Drug target
    Native mass spectrometry
    Tuberculosis
    Publication URI
    http://hdl.handle.net/10072/406982
    Collection
    • Theses - Higher Degree by Research

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