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Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT.

ABSTRACT: A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC¹²⁸⁰) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC₅₀ data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

SUBMITTER: Brown AS

PROVIDER: S-EPMC8309046 | biostudies-literature |

REPOSITORIES: biostudies-literature

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4'-Phosphopantetheinyl transferase PptT, a new drug target required for Mycobacterium tuberculosis growth and persistence in vivo.

Project description:The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4'-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.

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| S-EPMC9027781 | biostudies-literature

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis.

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| S-EPMC7021004 | biostudies-literature

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| S-EPMC3401038 | biostudies-literature

Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen.

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| S-EPMC5159837 | biostudies-literature

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