Chemical, Metabolic, and Cellular Characterization of a FtsZ Inhibitor Effective Against Burkholderia cenocepacia

Abstract

There is an urgent need for new antimicrobials to treat the opportunistic Gram-negative Burkholderia cenocepacia, which represents a problematic challenge for cystic fibrosis patients. Recently, a benzothiadiazole derivative, C109, was shown to be effective against the infections caused by B. cenocepacia and other Gram-negative and-positive bacteria. C109 has a promising cellular target, the cell division protein FtsZ, and a recently developed PEGylated formulation make it an attractive molecule to counteract Burkholderia infections. However, the ability of efflux pumps to extrude it out of the cell represents a limitation for its use. Here, more than 50 derivatives of C109 were synthesized and tested against Gram-negative species and the Gram-positive Staphylococcus aureus. In addition, their activity was evaluated on the purified FtsZ protein. The chemical, metabolic and cellular stability of C109 has been assayed using different biological systems, including quantitative single-cell imaging. However, no further improvement on C109 was achieved, and the role of efflux in resistance was further confirmed. Also, a novel nitroreductase that can inactivate the compound was characterized, but it does not appear to play a role in natural resistance. All these data allowed a deep characterization of the compound, which will contribute to a further improvement of its properties.

Antistaphylococcal Activity of the FtsZ Inhibitor C109

“…The compound was active at a concentration 128-fold lower than the MIC, and it greatly inhibited the formation of the bacterial biofilm in vitro at 0.125 μg/mL. These results showed increased activity of C109 in S. aureus biofilm inhibition compared to that previously reported against B. cenocepacia [ 24 , 25 ], confirming the great potentiality of the molecule against this microorganism [ 26 ]. Moreover, S. aureus biofilms were visualized by confocal laser scanning microscopy, highlighting significant differences in the biofilm structure of C109-treated cells compared to the untreated control.…”

“…It has been previously demonstrated that C109 targets the FtsZ protein of B. cenocepacia and of P. aeruginosa , blocking their GTPase and polymerization activities [ 24 , 26 ]. To assess C109 activity against the purified FtsZ of S. aureus , the wild type and a previously described more stable deleted version of the protein (12-316aa, ΔFtsZ) [ 31 ] were expressed and purified, as described in Section 4 .…”

“…To have a more complete overview of C109 antimicrobial activity, we also biochemically characterized the effect of the compound on its target at the molecular level. We previously identified the target of this broad-spectrum antibacterial in the Gram-negative rods B. cenocepacia and P. aeruginosa as the division protein FtsZ [ 24 , 26 ]. Indeed, C109 acts on the GTPase activity of the protein and prevents the polymerization [ 24 ].…”

“…Being considered a high-potential antimicrobial, an inhalable nanoformulation, with a great biofilm inhibition effect against B. cenocepacia , was developed for local treatment of CF lung infections [ 25 ]. Moreover, a deep characterization of C109 was performed, demonstrating that the benzothiadiazole molecular scaffold has a very good potential for the development of new, more potent antistaphylococcal compounds, since different chemically modified derivatives showed similar or even improved activity against S. aureus [ 26 ]. The great potentiality of this molecule is also given by its molecular target, the extremely conserved tubulin-like FtsZ protein [ 24 ].…”

“…The great potentiality of this molecule is also given by its molecular target, the extremely conserved tubulin-like FtsZ protein [ 24 ]. C109 inhibits the GTPase activity of B. cenocepacia and P. aeruginosa FtsZ, consequently blocking the formation of the polymers [ 24 , 26 ]. The ubiquity and the essential function of FtsZ make it an attractive drug target, as attested by the increasing number of FtsZ inhibitors characterized within the last few years [ 27 ].…”