Amsacrine Derivatives Selectively Inhibit Mycobacterial Topoisomerase I (TopA), Impair M. smegmatis Growth and Disturb Chromosome Replication

Abstract

Amsacrine, which inhibits eukaryotic type II topoisomerase via DNA intercalation and stabilization of the cleavable topoisomerase-DNA complex, promotes DNA damage and eventually cell death. Amsacrine has also been shown to inhibit structurally distinct bacterial type I topoisomerases (TopAs), including mycobacterial TopA, the only and essential topoisomerase I in Mycobacterium tuberculosis. Here, we describe the modifications of an amsacrine sulfonamide moiety that presumably interacts with mycobacterial TopA, which notably increased the enzyme inhibition and drug selectivity in vivo. To analyse the effects of amsacrine and its derivatives treatment on cell cycle, we used time-lapse fluorescence microscopy (TLMM) and fusion of the β-subunit of DNA polymerase III with enhanced green fluorescence protein (DnaN-EGFP). We determined that treatment with amsacrine and its derivatives increased the number of DnaN-EGFP complexes and/or prolonged the time of chromosome replication and cell cycle notably. The analysis of TopA depletion strain confirmed that lowering TopA level results in similar disturbances of chromosome replication. In summary, since TopA is crucial for mycobacterial cell viability, the compounds targeting the enzyme disturbed the cell cycle and thus may constitute a new class of anti-tuberculosis drugs.

SaccuFlow – A High-throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

“…Our screening results revealed a total of 18 compounds as potential inhibitors of sortase (Figure 5B). Of interest, amsacrine, which was previously reported to inhibit Mycobacterial topoisomerases 62 as well as D-alanylation of teichoic acids in S. aureus 63 , was identified as a potential inhibitor. We further tested the ability of amsacrine to inhibit sortase A in a titration assay, which showed a reduction in fluorescence levels in a concentration-dependent manner (Figure 5C).…”

Transcriptional Response of Streptomyces coelicolor to Rapid Chromosome Relaxation or Long-Term Supercoiling Imbalance

“…To date, there is little understood about the long-term effects of altered supercoiling, since most studied bacteria were able to quickly restore topological homeostasis. Moreover, the effects of increased negative supercoiling on global gene transcription in bacteria remain relatively unexplored, in part due to the limited availability of selective TopA inhibitors (Garcia et al, 2011; Cheng et al, 2013; Godbole et al, 2014; Szafran et al, 2018).…”

Watching DNA Replication Inhibitors in Action: Exploiting Time-Lapse Microfluidic Microscopy as a Tool for Target-Drug Interaction Studies in Mycobacterium

“…The growth rate (optical density at 600 nm per minute) was estimated by analyzing the slope of the linearly fitted correlation in the exponential growth phase. The percentage of growth inhibition was calculated by comparing the growth rates obtained in the presence of the tested antibiotics to the growth rate obtained without any inhibitor (which was defined as 100%), as described previously (78). The IC 50 was calculated for each strain from the inhibition curve plotted using the R package software and was taken as the concentration of a particular compound that inhibited the cell growth rate by 50%.…”

“…TLMM was performed as previously described (47, 78) using B04A plates with an Onix flow-control system (Merck-Millipore). Cells loaded into the observation chamber were exposed to fresh 7H9-OADC-Tween 80 for 5 h, 7H9-OADC-Tween 80-inhibitor for 5 h, and fresh 7H9-OADC-Tween 80 without antibiotic for 7 h. All experiments were performed under continuous pressure (1.5 lb/in 2 ) at 37°C.…”