Harvard University chemist Daniel Kahne has spent much of his career studying the fundamentals of how bacteria thrive and evade attack.
His lab has a special interest in gram-negative bacteria, which have an outer membrane that many antibiotics cannot cross.
Among these bacteria is carbapenem-resistant Acinetobacter baumannii, or CRAB. Designated by the World Health Organization as a “critical priority” for antibiotic development, CRAB kills hundreds of critically ill patients in the U.S. each year, usually in hospital settings, by causing untreatable blood, lung, or urinary tract infections.
Strengthened by partnerships with basic research labs and the pharmaceutical industry, Kahne and colleagues are working to usher in a new class of antibiotics to combat the A. baumannii superbug — and perhaps others.
Such an achievement would have significant benefit for human health. No new antibiotics have been introduced for gram-negative bacteria in more than 50 years, and the WHO has declared that development of new antibiotics remains “inadequate” to address the global threat of antibiotic resistance.
Looking for a way through the membrane
The question of how A. baumannii and other gram-negative bacteria construct their second, outer membrane has occupied Kahne, the Higgins Professor of Chemistry and Chemical Biology at Harvard University and professor of biological chemistry and molecular pharmacology in the Blavatnik Institute at Harvard Medical School, for the past 25 years.
“There are a set of protein machines that are conserved in all gram-negative bacteria that make this membrane,” he explained, “and so we study each of these machines.”
The membranes are dotted with large fat-and-carbohydrate molecules called lipopolysaccharides (LPS) that act as protective armor.
Bacteria assemble LPS molecules inside their cytoplasm before moving them to their outer membranes. Kahne wanted to know how the bacteria orchestrate this transport, in part because scientists had long suspected that the multistep process could provide new targets for future antibiotics.
In 2010 Kahne’s team was the first to propose a mechanism for how gram-negative bacteria manage LPS transport.
The researchers showed that LPS molecules use a “trans-envelope bridge” made of seven different proteins to travel from the cytoplasm across the inner cell membrane, through an aqueous space called the periplasmic compartment, and across the outer membrane, where the bacterium pushes the LPS molecules out one at a time like a Pez dispenser.
The team published several more studies supporting their bridge model. In 2018 they reconstituted the bridge from pure proteins in lab dishes. In November 2023 they showed the bridges forming and transporting LPS in live cells.
Probing a potential new antibiotic
Meanwhile, the international biotechnology company Roche had been working to develop a new type of antibiotic effective against CRAB infections.
The company had genetic evidence to believe one of the potential antibiotics killed A. baumannii by disrupting the LPS transport machinery Kahne’s lab studies. No other drug on the market currently aims to kill bacteria via that machinery.
Roche approached the Harvard researchers in late 2020 to confirm what the compound, called zosurabalpin, was doing.
“Dan is a well-recognized world leader in this field, and we saw the potential for synergy between the two teams in order to validate the target and mechanism of action of zosurabalpin as well as gain further insight into the fundamental biological process of LPS transport,” said Kenneth Bradley, head of infectious diseases discovery at Roche Pharma Research and Early Development.
Kahne’s group teamed up with Roche scientists and the lab of Andrew Kruse, professor of biological chemistry and molecular pharmacology in the Blavatnik Institute at HMS. They soon had their answer.
In two Nature papers in January 2024, they reported that Roche’s new class of antibiotics acts on the LPS transport mechanism and provided details of how zosurabalpin does so.
“It was very exciting to be able to use all of the tools that the students in my group have developed over the last two decades to quickly figure out the mechanism of action of Roche’s compound,” Kahne said.
What it took to see how the compound works
The tools included structural, biochemical, and genetic approaches to solve complex protein structures.
Kahne lab postdoctoral fellow Karanbir Pahil developed a procedure for producing large quantities of pure Acinetobacter transport proteins. The research uncovered the first direct evidence that zosurabalpin interferes with LPS molecules.
Kruse’s team, including Morgan Gilman, an HHMI Hanna Gray Fellow in Biological Chemistry and Molecular Pharmacology at HMS, applied their expertise in cryo-electron microscopy to capture the molecular details. Cryo-EM is particularly helpful for studying dynamic proteins that can twist into multiple shapes.
“This very complicated transporter clearly falls into that category,” Kruse said.
The work revealed the LPS molecule’s structure as it binds to zosurabalpin.
The Kahne and Kruse labs then showed that zosurabalpin disrupts the transport of LPS to the cell surface by binding to a pocket that encompasses part of the protein bridge as well as the LPS molecule itself. The effort involved solving seven protein structures.
The researchers noted that it’s unusual for a drug to bind to a composite surface created by a protein machine and the cellular molecule it acts on.
They found that the compound causes LPS to accumulate in the wrong part of the bacterium — jamming LPS inside its transporter, weakening the cell’s membrane, and eventually destroying the cell.
What could happen next
Zosurabalpin is now in phase 1 clinical trials.
If approved by the U.S. Food and Drug Administration, it would be the first new treatment for A. baumannii infections in half a century.
It would also be the first antibiotic to target the LPS transport mechanism. By contrast, most widely used antibiotics, including penicillin and carbapenem, work by inhibiting synthesis of the bacterial cell wall. Other antibiotic types disrupt ribosomes and protein synthesis.
The pocket that zosurabalpin binds to is specific to A. baumannii, but the researchers note that a similar pocket exists in other gram-negative bacteria, including E. coli. They are excited by the possibility that the compound, if successful, could be modified to treat other pathogens.
“We are starting to have a fairly deep understanding of how many of these molecular machines work,” Kruse said. “I think that’s going to be incredibly helpful in designing next-generation antibiotics.”
The Harvard Office of Technology Development spearheaded the three-year sponsored research agreement between Roche and the Kahne lab that resulted in the pair of Nature papers.
via HMS News
