An anti-tuberculosis compound screen using a zebrafish infection model identifies an aspartyl-tRNA synthetase inhibitor

Abstract

Finding new anti-tuberculosis compounds with convincing in vivo activity is an ongoing global challenge to fight the emergence of multi-drug resistant Mycobacterium tuberculosis isolates. In this work, we exploited the medium-throughput capabilities of the zebrafish embryo infection model with Mycobacterium marinum as a surrogate for M. tuberculosis. Using a representative set of clinically established drugs, we demonstrate that this model could be predictive and selective for antibiotics that can be administered orally. We further used the zebrafish-infection model to screen 240 compounds from an anti-TB hit library for their in vivo activity and identified 14 highly active compounds. One of the most active compounds was the tetracyclic compound TBA161, which was studied in more detail. Analysis of resistant mutants revealed point mutations in aspS (rv2572c), encoding an aspartyl-tRNA synthetase. The target was genetically confirmed, and molecular docking studies propose possible binding of TBA161 in a pocket adjacent to the catalytic site. This study showed that the zebrafish-infection model is suitable to rapidly identify promising scaffolds with in vivo activity.

Modulating mycobacterial envelope integrity for antibiotic synergy with benzothiazoles

“…To investigate the effect of the benzothiazoles on mycobacteria during infection, we used the previously described M. marinum –zebrafish embryo infection model ( Habjan et al, 2021 ). Zebrafish embryos were infected via yolk injection with M. marinum , expressing a red fluorescent protein ( tdTomato ).…”

“…The compound’s intracellular activity was investigated in macrophage infection studies, as described previously ( Habjan et al, 2021 ). Infection stocks of M. tuberculosis H37Rv transformed with pTetDuo were prepared in RPMI GlutaMAX with 10% FBS as the infection medium, supplemented with 20% glycerol, and stored at −80°C.…”

“…Images of each well were taken at specific wavelengths (excitation/emission: 385/455 nm; 470/519 nm; 550/610 nm). Images were analyzed using a custom-made pipeline in CellProfiler 3.19 (Broad Institute, Cambridge, USA), as described previously ( Habjan et al, 2021 ). Briefly, the fluorescent signal of the ATc-inducible GFP around each macrophage was used as a readout for viable intracellular bacteria in each macrophage, whereas the number of stained and detected nuclei was used as a readout for the number of macrophages in each treatment group.…”

Diving into drug-screening: zebrafish embryos as an in vivo platform for antimicrobial drug discovery and assessment

“…Moreover, nine out of these 11 belong to existing classes of antibiotics to which resistance mechanisms could already be present in clinical isolates (WHO 2020 ). One of the bottlenecks in introducing new compounds is that the in vitro data does not always translate to the compound’s efficacy in animal models or into favorable clinical outcomes (Aspatwar et al 2017 , Habjan et al 2021 , Schouten et al 2022 ). Clearly, there is a need for preclinical models with a higher translational prediction value.…”

“…In some cases, e.g. when using Mycobacterium marinum , the model allows for automated and robotized infection step, thus increasing experimental throughput by enabling testing many compounds (Spaink et al 2013 , Habjan et al 2021 ).…”