Synthesis and mechanism-of-action of a novel synthetic antibiotic based on a dendritic system with bow-tie topology

Abstract

Over the course of the last decades, the continuous exposure of bacteria to antibiotics—at least in parts due to misprescription, misuse, and misdosing—has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent.

The Novel Synthetic Antibiotic BDTL049 Based on a Dendritic System Induces Lipid Domain Formation while Escaping the Cell Envelope Stress Resistance Determinants

“…BDTL100 was used in fluorescent microscopy localization studies, which revealed its accumulation at the cytoplasmic membrane of the bacterial cells leading to lipid domain formation ( Figure 3 ), and supported our previous observations pointing toward a heterogeneous affectation of the bacterial cell population. Interestingly, even though BDTL049 induces a strong cell envelope stress response in B. subtilis (as shown by previous transcriptomic analysis [ 18 ]), the sensitivity against BDTL049 of several mutant strains revealed that, except for a minor contribution of the Dlt system ( Figure 4 ), the resistance determinants which mediate the cell envelope stress response to other antibiotics are unable to mount an effective resistance response against the novel compound leaving the cell rather defenseless. Finally, the biocompatibility of BDTL049 was investigated by determining its cytotoxicity in human cell lines.…”

“…Our previous results indicated that the cytoplasmic membrane is the primary cellular target of the novel antimicrobial compound BDTL049, which depolarizes the membrane and leads to pore formation [ 18 ]. We next aimed at visualizing the interaction of the compound with the bacterial cell in vivo.…”

“…The dendritic compounds used in this work ( Figure 1 ) were synthesized following a similar (BDTL049) [ 18 ] or adapted (BDTL100) [ 19 ] protocol described previously. For BDTL100 (131.1 mg; 0.131 mmol), the thiol-ene reaction was carried out with two thiol derivatives.…”

“…All the sensitivity experiments for determination of the Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were performed in Mueller-Hinton (MH) Broth (Carl Roth, Karlsruhe, Germany), as previously described [ 18 ]. The experiments were performed with at least three biological replicas on different days in the case of the wild-type strain and in biological duplicates (two clones) and technical triplicates (on different days) in the case of the mutant strains.…”

“…We recently reported de novo rational design, the in vitro chemical synthesis, and the in vivo experimental testing of the new antimicrobial agent termed BDTL049 [ 18 ]. Based on a first-generation carbosilane scaffold, it is a bow-tie topology ammonium-terminated dendrimer with a hydrophobic inner skeleton but a positively charged periphery.…”