THP-1 and Dictyostelium Infection Models for Screening and Characterization of Anti-Mycobacterium abscessus Hit Compounds

Abstract

!!NCR1!! presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. M. abscessus is able to grow intracellularly in human macrophages, suggesting that intracellular models can facilitate drug discovery. Thus, we have developed two host cell models: human macrophages for use in a new high-content screening method for M. abscessus growth and a Dictyostelium discoideum infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular M. abscessus led to the identification of two hit compounds with distinct growth inhibition. A collection of 317 human kinase inhibitors was analyzed, with the results yielding three compounds with an inhibitory effect on mycobacterial growth, strengthening the notion that host-directed therapy can be applied for M. abscessus.

High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis

“…The total circle spot intensity of each well corresponds to the intracellular bacterial load. We (Richter et al, 2019) and others have previously validated these fluorescent measurements closely correspond with CFUs. Nuclei stain (Hoechst 33342) was used to quantify THP-1 cell loss (due to cytotoxicity or loss of adherence).…”

A Screening of the MMV Pandemic Response Box Reveals Epetraborole as A New Potent Inhibitor against Mycobacterium abscessus

“…Besides, Mab is an intracellular pathogen. During infection, Mab is engulfed by human macrophages, overcomes a host’s immune defense mechanisms, replicates, and resides inside human immune cells [ 15 ]. Therefore, only compounds with a high enough penetration ability to pierce the macrophage plasma membrane and the thick cell membrane of the Mab will lead to an inhibition of intracellular mycobacterial growth.…”

Advances in host‐based screening for compounds with intracellular anti‐mycobacterial activity

“…While these screens used large-scale unbiased compound libraries, other groups have adapted an approach of using smaller focused An interesting aspect of this study was the use of two different infection models. In addition to THP-1, the authors also used the amoeba Dictyostelium discoideum to confirm one of the hit compounds (Richter et al, 2019). Such confirmatory approaches in orthogonal models are important to identify conserved host cell-type independent targets.…”

“…The recent development of a method to infect zebrafish larvae by incubating in media containing M. marinum (Dalton et al, 2017), or robotic injection (Spaink et al, 2013), if adapted successfully to a screening format with good assay metrics and reproducibility could alleviate the need for manual injection of individual larvae. Finally, the amoebae model (Hanna et al, 2020;Ouertatani-Sakouhi et al, 2017;Richter et al, 2019;Trofimov et al, 2018) that involves infection of single celled ameobae with either M. marinum or M. tuberculosis infection offers a neat host-pathogen interaction space that combines the advantages of in vitro platform and relevance of an in vivo system. Cytotoxicity assays performed in amoeba model compares favourably with the mammalian cells (Trofimov et al, 2018).…”