A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics

Anthony D Verderosa,Begoña Heras,Rabeb Dhouib,Yaoqin Hong,Taylah K Anderson,Makrina Totsika

doi:10.1038/s41598-021-81007-y

Anthony D Verderosa, Begoña Heras + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-81007-y

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Abstract

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors.

Highlights

In 2014 the World Health Organisation (WHO) released a statement declaring antimicrobial resistance (AMR) as a public health priority that demands decisive global action[1]
For monitoring DsbA function in vivo, the bacterial motility assay on soft agar has been most commonly used[27,28,29] and more recently this method was applied to DsbA inhibitor testing in vivo[12,30]
As the ASST gene in uropathogenic Escherichia coli (UPEC) is not expressed under standard growth conditions[29] and in order to make our assay transferrable to non ASST-encoding bacteria, we chose to produce ASST from a plasmid vector previously shown to depend on DsbA for activity in UPEC and S

Summary

Introduction

In 2014 the World Health Organisation (WHO) released a statement declaring antimicrobial resistance (AMR) as a public health priority that demands decisive global action[1]. We develop a pipeline of robust cell-based assays for the in vivo evaluation of inhibitors against the DsbA antivirulence target. For monitoring DsbA function in vivo, the bacterial motility assay on soft agar has been most commonly used[27,28,29] and more recently this method was applied to DsbA inhibitor testing in vivo[12,30]. In many pathogens, such as uropathogenic Escherichia coli (UPEC), and Salmonella enterica serovar Typhimurium We present a comprehensive pipeline of cell-based assays that provide an accurate and high throughput platform for the identification of DsbA inhibitors and their subsequent development from hits to leads, and from lead optimisation to early preclinical candidate validation

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