In the innermost layer of a parasitic worm sits the starting material for what may become a powerful new antibiotic. Darobactin — a natural compound produced by bioluminescent bacteria in the guts of these worms — was discovered by Kim Lewis’s team at Northeastern University in 2019 (1).

While the original darobactin molecule showed activity against gram-negative bacteria in vitro and in mouse models, Rolf Müller and his lab members at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) have been creating optimized analogs of the antibiotic (2). “One of the front runners that we discovered among 80 compounds is D22,” said Andreas Kany, a postdoctoral researcher at HIPS. “We wanted to take this compound to the next level and perform in vivo characterization.”

In a new study, Kany and his colleagues tested the activity of D22 in multiple animal models (3). “What we found is that darobactin D22 is active in diverse models of infection, in both mice and zebrafish, and across different gram-negative bacteria,” said Kany, who was also the lead author of the paper.

The World Health Organization designated gram-negative bacteria including Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriales as priority pathogens to target for antibiotic development. Darobactins act on BamA, a transmembrane protein in the outer membrane of gram-negative bacteria, where they interfere with the folding and insertion of proteins there. This feature makes darobactins promising as antibiotics because they only need to cross one of gram-negative bacteria’s two membranes to kill the cell.

The researchers began by infecting zebrafish embryos with A. baumannii as mouse models for A. baumannii are challenging. They found that darobactin D22 and D69 — another derivative they developed — completely cleared the infection. This was the first time a darobactin proved effective against A. baumannii   in an in vivo  model.

When they turned to mouse models, they began by assessing D22’s pharmacokinetics and found that its half-life was less than one hour. Tobramycin, another antibiotic used to treat gram-negative bacterial infections, has a serum half-life of about two hours (4). “There are actually antibiotics on the market which have relatively low half-life. It just means that the compound needs to be dosed more often,” said Kany. Still, Kany and his colleagues plan to increase the compound’s half-life in further optimizations.

The researchers also saw that D22 reached many locations where bacterial infections can occur, so they used this finding to model soft tissue infections, kidney infections, and urinary tract infections (UTIs).

In mice infected with P. aeruginosa in their thighs, the researchers noted a statistically significant reduction in the bacterial burden with D22 treatment compared to those without D22 treatment. In an Escherichia coli  UTI model, they found that D22 reduced the number of bacteria in the urine, bladder, and kidneys. Kany said that for the darobactin compound scaffold, “it’s the first UTI efficacy data out there.”

This is a really interesting drug because we are desperate for antibiotics for gram-negative bacteria. 
- Lori Burrows, McMaster University

Kany and his colleagues then took D22 one step further. To model a severe infection, they looked to a mouse model of peritonitis, an infection in the abdominal cavity that can lead to sepsis. Here, they found that four doses of intravenous or subcutaneous D22 administration led to 100 percent survival and that the infection fully cleared the blood.

These types of experiments help move drug candidates further along the antibiotic development pipeline, said Lori Burrows, a microbiologist at McMaster University who was not involved in this study.

“I am excited that there are some new drugs that are targeting new targets,” she added because, she mentioned, most antibiotics focus on a limited number of mechanisms. “This is a really interesting drug because we are desperate for antibiotics for gram-negative bacteria.”

Because of the positive results of their in vivo study, Kany and his colleagues hope to move D22 forward in the development process. “We consider D22 a lead,” he said. “It could be ready for lead optimization.”

References

1. Imai, Y. et al. A new antibiotic selectively kills gram-negative pathogens. Nature  576, 459-464 (2019).
2. Seyfert, C.E. et al. New genetically engineered derivatives of antibacterial darobactins underpin their potential for antibiotic development. J Med Chem   66, 16330-16341 (2023).
3. Kany, A.M. et al. In vivo  activity profiling of biosynthetic darobactin D22 against critical gram-negative pathogens. ACS Infect Dis  10, 4337-4346 (2024).
4. Reyhanoglu, G. & Reddivari, A.K.R. Tobramycin. StatPearls [Internet] (2023).