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

Alistair S Brown,James Jung,Jeremy G Owen,David F Ackerley,Edward N Baker

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, 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.

Highlights

Despite diminishing rates of infection, the disease burden of Mycobacterium tuberculosis remains high, with 2019 seeing approximately 10 million people infected and 1.4 million deaths worldwide [1]
To determine whether BpsA could be used to screen for inhibitors of PptT, it was first necessary to show that PptT is capable of activating BpsA from the inactive apo to active holo form
PptT was capable of rapidly activating apo-BpsA in vitro, yielding a typical indigoidine synthesis curve that reflects an initial burst of indigoidine synthesis followed by subsequent indigoidine precipitation [22] (Figure 1C)

Summary

Introduction

Despite diminishing rates of infection, the disease burden of Mycobacterium tuberculosis remains high, with 2019 seeing approximately 10 million people infected and 1.4 million deaths worldwide [1]. Mega-synthetases, including the fatty acid synthetase (FAS) I and II systems and polyketide synthetases (PKSs), play crucial roles in the biosynthesis of these lipids [6]. A further megasynthetase family, the non-ribosomal peptide synthetases (NRPSs), is required to produce the important virulence factor mycobactin [7]. Each of these mega-synthetases requires the attachment of a phosphopantetheinyl (Ppant) arm to one or more carrier protein (CP)

Methods

Results

Conclusion