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A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors

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    A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors
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    † Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, via Ferrata 9, 27100 Pavia, Italy
    ‡ Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina CH1, 84215 Bratislava, Slovakia
    ⊥ Department of Molecular Medicine, University of Padova, via Gabelli 63, 35121 Padova, Italy
    § Diseases of the Developing World, GlaxoSmithKline, Calle Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
    ∥ Collaborative Drug Discovery Inc., 1633 Bayshore Highway, Suite 342, Burlingame, California 94010, United States
    *L.R.C. E-mail: [email protected]
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    ACS Infectious Diseases

    Cite this: ACS Infect. Dis. 2017, 3, 6, 428–437
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    https://doi.org/10.1021/acsinfecdis.7b00006
    Published May 5, 2017
    Copyright © 2017 American Chemical Society
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    Abstract

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    Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with Ki values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

    Copyright © 2017 American Chemical Society

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.7b00006.

    • Effects of GSK compounds against different ATP binding enzymes, MIC values of M. tuberculosis wild-type and pyrG mutant strains to GSK compounds, ratios of radioactivity incorporated to [14C]UTP and [14C]CTP of the control experiments, PyrG kinetic analysis toward UTP in the presence compounds, effects of the GSK compounds against PyrG glutaminase activity, SDS-PAGE of the purification steps of HuCTPS-1, IC50 values of compounds to the wild-type and the V186G mutant PyrG enzyme, and characterization of the pyrG conditional mutant TB456 (PDF)

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    ACS Infectious Diseases

    Cite this: ACS Infect. Dis. 2017, 3, 6, 428–437
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acsinfecdis.7b00006
    Published May 5, 2017
    Copyright © 2017 American Chemical Society
    Request reuse permissions

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