Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening

Hasan Tükenmez,Mikael Elofsson,Isabel Edström,Stina Berglund Fick,Christer Larsson,Ramesh Ummanni,Charlotta Sundin

doi:10.1038/s41598-018-37176-4

Hasan Tükenmez, Mikael Elofsson + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-37176-4

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Abstract

High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but also virulence inhibitors with either bacterial or host cell targets, an assay monitoring lung fibroblast survival upon infection was developed and optimized for 384-plate format and robotic liquid handling. By using Mycobacterium marinum as surrogate organism, 28,000 compounds were screened at biosafety level 2 classification, resulting in 49 primary hits. Exclusion of substances with unfavourable properties and known antimicrobials resulted in 11 validated hits of which 7 had virulence inhibiting properties and one had bactericidal effect also in wild type Mycobacterium tuberculosis. This strategy to discover virulence inhibitors using a model organism in high-throughput screening can be a valuable tool for other researchers working on drug discovery against tuberculosis and other biosafety level 3 infectious agents.

Highlights

Drug resistant tuberculosis (TB) is a major health threat and the World Health Organization (WHO) estimates that 490,000 new cases of multidrug-resistant TB (MDR-TB) occurred in 20161
Since High-throughput screening (HTS) is highly dependent on advanced liquid handling robots and analytical instruments, it is generally inaccessible to drug discovery endeavours using wild type (WT) M. tuberculosis or other biosafety level 3 (BSL3) pathogens
To develop a HTS method for discovery of antimicrobials, but any type of compound having an adverse effect on mycobacterial virulence, we took advantage of the cytolytic properties present in M. marinum

Summary

Introduction

Drug resistant tuberculosis (TB) is a major health threat and the World Health Organization (WHO) estimates that 490,000 new cases of multidrug-resistant TB (MDR-TB) occurred in 20161. Few institutions are capable of performing HTS under BSL3 conditions and time on the instruments is regularly scarce and costly This constitutes a major bottleneck in the search for new drugs against TB in general and in discovery of virulence inhibitors in particular since attenuated strains such as M. bovis Bacille Calmette-Guerin (BCG) or M. tuberculosis H37Ra is of limited use. Parts of our team have previously discovered and further developed virulence inhibitors of type III secretion in Yersinia pseudotuberculosis and Chlamydia trachomatis[13,14,15]. To circumvent the need of a BSL3 screening facility, we have developed a HTS strategy to discover mycobacterial virulence inhibitors using the fish and frog pathogen Mycobacterium marinum as a model organism for M. tuberculosis. We screened 28,000 chemical compounds with M. marinum under BSL2 conditions and discovered 11 novel hits as potential mycobacterial virulence inhibitors. The HTS strategy developed in this study can be a valuable tool in TB drug discovery, as it provides the opportunity to screen for virulence inhibitors under BSL2 conditions

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