Alexander Fleming’s discovery of penicillin in 1928 is one of the most famous examples of serendipity in modern science. The pioneering microbiologist returned from vacation to find that blue-green mold had colonized a used Petri dish and decimated its bacterial neighbors (1). Within 15 years, chemists managed to identify, purify, and mass produce the molecule that became the world’s first and most famous antibiotic. Now, researchers seeking to develop new antibiotics, like Roche infectious disease scientist Kenneth Bradley, start off with a much deeper understanding of bacteria and their weaknesses, but it turns out there’s still a great deal of chance involved in the process.
In a pair of Nature studies, Bradley and his colleagues detailed the exploratory screens and open-ended experiments that enabled them to identify a synthetic molecule capable of killing a drug-resistant strain of infectious Acinetobacter baumannii bacteria (2,3). They found that the new antibiotic relies on a unique mechanism of action that human pathogens have not yet encountered in the age of modern medicine.
“If it makes it through all of clinical development to launch, it would be potentially the first in over 50 years to be a novel class for treating Acinetobacter,” said Bradley.
According to Bradley, it all began about 10 years ago when Roche acquired a chemical library consisting of molecules in the macrocyclic peptide (MCP) class. MCP molecules are artificial and relatively easy to synthesize, but they have more structural complexity than a lot of the small molecules that make up the pharmaceutical screening libraries that Bradley is used to seeing in drug development scenarios (4).
“Existing commercial antibiotics typically are derived from natural products,” said Bradley. “The properties that they have are different than the smaller, more simple molecules that we typically use as starting points.”
He and his team hypothesized that the complex nature of MCP molecules would make them more capable of killing A. baumannii and other gram-negative bacteria, which have two membranes that can exclude simpler synthetic molecules.
The scientists exposed drug resistant A. baumannii colonies to each of the 44,985 MCP molecules in their chemical library. Of all the MCP molecules screened, they identified one that killed A. baumannii at the lowest concentration. Bradley and his team tinkered with the molecule’s structure to see if they could improve its potency and selectivity for A. baumannii. Finally, they produced an MCP that looked like a preclinical antibiotic.
Killing cultured bacteria is not the same as killing bacteria growing in a living body, so Bradley and his team injected it into mice with established drug resistant A. baumannii infections.
“We were quite pleased to see that it actually did reduce the bacteria,” said Bradley. “Unfortunately, when we then administered that same compound intravenously, which would be the route of administration in humans in a hospital, we saw some safety flags.”
Some of the rats that received the molecule intravenously died, and bloodwork showed that the molecule caused fats to precipitate out of the animals’ blood. Bradley’s team realized that their molecule was much more attracted to lipids than typical antibiotics, so they modified it again. The more hydrophilic version, which they named zosurabalpin, turned out to be much safer for the rats.
But Bradley and the rest of the researchers still weren’t satisfied. They wanted to know how it worked, so they used evolution to figure it out.
“By applying an external pressure — in this case, gradually increasing the concentration of the antibiotic — you could select for mutations that were resistant, and those mutations may or may not map back to the target of the antibiotic,” said Bradley. “In this case, we were able to identify a smoking gun.”
All the bacteria that managed to survive the onslaught of zosurabalpin had mutations in a complex of proteins that transport lipopolysaccharide (LPS) molecules from a bacterium’s inner membrane to its outer membrane, where LPS molecules help protect the organism from the outside world.
If it makes it through all of clinical development to launch, it would be potentially the first in over 50 years to be a novel class for treating Acinetobacter.
– Kenneth Bradley, Roche
Even though the researchers had found a smoking gun, they weren’t sure they knew why it had been fired. “Sometimes mutations will pop up that allow bacteria to spike but don’t actually represent the specific target pathway that the molecule is actually killing,” said Bradley. “It could be a compensatory pathway.”
The Roche scientists enlisted the help of Harvard University chemist Daniel Kahne and his research group to visualize zosurabalpin interacting with bacterial proteins. The Harvard chemists used cryogenic electron microscopy to investigate, and they found that zosurabalpin binds LPS within the Acinetobacter transporter protein. “It’s not very often that you find a compound that can actually bind to the substrate of a transporter and, in essence, jam it up,” said Bradley. “That was a really interesting insight for us.”
Since publishing their Nature studies introducing zosurabalpin, Bradley and the Roche team have moved the drug into clinical trials to test its safety in humans. Julio Camarero, a chemist at the University of Southern California who was not involved with the study, is excited to see what comes next for zosurabalpin. He was impressed by the drug’s effectiveness against A. baumannii bacteria, but he wondered if the drug’s high specificity would limit its future applications.
“It is a good thing if you want to kill those bacteria, but if you want to design a broad-spectrum antibacterial, then it’s not,” said Camarero. Zosurabalpin binds LPS in the transporter protein of Acinetobacter bacteria, but not in the transporter proteins of other gram-negative bacteria, like Escherichia coli and Pseudomonas species.
For his part, Bradley sees zosurabalpin’s specificity as an opportunity, rather than a limitation. He said, “That actually opens up the possibility for us to think about blocking this pathway in other species.”
References
- Ligon, B. Penicillin: its discovery and early development. Semin Pediatr Infect Dis 15, 52-57 (2004).
- Zampaloni, C. et al. A novel antibiotic class targeting the lipopolysaccharide transporter. Nature 625, 566-571 (2024).
- Pahil, K. et al. A new antibiotic traps lipopolysaccharide in its intermembrane transporter. Nature 625, 572-577 (2024).
- Vinogradov, A. et al. Macrocyclic peptides as drug candidates: recent progress and remaining challenges. J Am Chem Soc 141, 4167-4181 (2019).