Browsing by Author "Kenyon, C. P."

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    Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform
    (Public Library of Science, 2017-03-29) Theron, A.; Roth, R. L.; Hoppe, H.; Parkinson, C.; Van Der Westhuyzen, C. W.; Stoychev, S.; Wiid, I.; Pietersen, R. D.; Baker, B.; Kenyon, C. P.
    ENGLISH ABSTRACT: Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.
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    Novel kinase platform for the validation of the anti-tubercular activities of Pelargonium sidoides (Geraniaceae)
    (BMC (part of Springer Nature), 2020-09-29) Lukman, V.; Odeyemi, S. W.; Roth, R. L.; Mbabala, L.; Tshililo, N.; Vlok, N. M.; Dewar, M. J. B.; Kenyon, C. P.
    Background: Pelargonium sidoides is an important traditional medicine in South Africa with a well-defined history of both traditional and documented use of an aqueous-ethanolic formulation of the roots of P. sidoides (EPs 7630), which is successfully employed for the treatment of respiratory tract infections. There is also historical evidence of use in the treatment of tuberculosis. The aim of this study was to develop a platform of Mycobacterium tuberculosis (Mtb) kinase enzymes that may be used for the identification of therapeutically relevant ethnobotanical extracts that will allow drug target identification, as well as the subsequent isolation of the active compounds. Results: Mtb kinases, Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase were cloned, produced in Escherichia coli and characterized. HPLC-based assays were used to determine the enzyme activities and subsequently the inhibitory potentials of varying concentrations of a P. sidoides extract against the produced enzymes. The enzyme activity assays indicated that these enzymes were active at low ATP concentrations. The 50% inhibitory concentration (IC50) of an aqueous root extract of P. sidoides against the kinases indicated SK has an IC50 of 1.2 μg/ml and GK 1.4 μg/ml. These enzyme targets were further assessed for compound identification from the P. sidoides literature. Conclusion: This study suggests P. sidoides is potentially a source of anti-tubercular compounds and the Mtb kinase platform has significant potential as a tool for the subsequent screening of P. sidoides extracts and plant extracts in general, for compound identification and elaboration by selected extract target inhibitor profiling.