Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 2 June 2021 / Approved: 3 June 2021 / Online: 3 June 2021 (13:22:33 CEST)

A peer-reviewed article of this Preprint also exists.

Brown, A.S.; Owen, J.G.; Jung, J.; Baker, E.N.; Ackerley, D.F. Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT. Pharmaceutics 2021, 13, 1066. Brown, A.S.; Owen, J.G.; Jung, J.; Baker, E.N.; Ackerley, D.F. Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT. Pharmaceutics 2021, 13, 1066.

Journal reference: Pharmaceutics 2021, 13, 1066
DOI: 10.3390/pharmaceutics13071066

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, colorimetric 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.

Keywords

PPTase; NRPS; indigoidine; PptT; antibiotic screening

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