A team of Princeton researchers led by Prof. Zemer Gitai have found an antibiotic that can simultaneously puncture bacterial walls and destroy folate within their cells -- taking out even monstrous bacteria with the effectiveness of a poisoned arrow -- while proving immune to antibiotic resistance. [Matilda Luk, Princeton University Office of Communications]

A team of Princeton researchers has identified a compound that can kill both Gram-positive and Gram-negative bacteria via two independent mechanisms, as well as resist antibiotic resistance. The compound, designated SCH-79797, works by simultaneously targeting bacterial folate metabolism and membrane integrity. When tested in vivo, SCH-79797 was found to be more effective than a combination of existing treatments against methicillin-resistant Staphylococcus aureus. A more potent derivative of SCH-79797, called Irresistin-16, was also found to be effective against Neisseria gonorrhoeae in a mouse vaginal infection model.

“This is the first antibiotic that can target Gram-positives and Gram-negatives without resistance,” said Zemer Gitai, PhD, Princeton’s Edwin Grant Conklin professor of biology, and senior author of the team’s published paper in Cell. “From a ‘why it’s useful’ perspective, that’s the crux. But what we’re most excited about as scientists is something we’ve discovered about how this antibiotic works—attacking via two different mechanisms within one molecule—that we are hoping is generalizable, leading to better antibiotics—and new types of antibiotics—in the future.” Gitai and colleagues described their research in a paper titled, “A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.”

Pathogenic bacteria can be classified as either Gram-positive or Gram-negative, after the scientist Hans Christian Gram, who developed a staining technique that can distinguish between them. The key difference is that Gram-negative bacteria are protected by an outer layer that resists most antibiotics. In fact, no new classes of Gram negative-killing drugs have come to market in nearly 30 years. “ … only six new classes of antibiotics have been approved in the past 20 years, none of which are active against Gram-negative bacteria,” the authors wrote. And despite recent efforts to “reinvigorate antibiotics research,” they continued, most of this work has resulted in compounds that function via similar mechanisms to those of traditional antibiotics, while recently discovered natural product-derived candidates have either only been effective against Gram-positive bacteria, or are prone to resistance.

“Thus, there is still a strong need for characterizing new classes of antibiotics with distinct mechanisms of action (MoA), especially those that target Gram-negatives with low resistance frequency,” the team noted. “An ideal antibiotic would be hard to develop resistance against, able to kill both Gram-positive and Gram-negative bacteria, and easy to access.” The holy grail of antibiotics research would thus be an antibiotic that is effective against the pathogen, is immune to resistance, and is safe in humans.

A team of Princeton researchers led by  Zemer Gitai, PhD, have found an antibiotic that can simultaneously puncture bacterial walls and destroy folate within their cells—taking out even monstrous bacteria with the effectiveness of a poisoned arrow—while proving immune to antibiotic resistance. Authors on the new paper in Cell include Joseph Sheehan (left), Gabriel Moore (4th from left, in blue), Sophia Hsin-Jung Li (5th from left, in pink), James Martin (4th from right, in baseball cap), Zemer Gitai (2nd from right), and Benjamin Bratton (right), seen here on a pre-social-distancing walk across the Princeton campus. [Courtesy of Zemer Gitai]
The Princeton University team identified the new candidate, SCH-79797, as one of the most potent “hits” in a small molecule library screen. Interestingly, the compound had previously been reported as a human PAR-1 antagonist. Another study had suggested that SCH-79797 increased the ability of neutrophils to kill bacteria, perhaps by directly functioning as an antibiotic. Studies focusing on its anticoagulant activity had also suggested that SCH-79797 could be safely tolerated in animals, so the Princeton researchers carried out further studies to further investigate its antibiotic properties.

Bacteria are adept at evolving quickly to resist antibiotics, but the researchers found that they were unable to generate any resistance to SCH-79797. For an antibiotics researcher, this is like discovering the formula to convert lead to gold, or riding a unicorn—something everyone wants but no one really believes exists, said co-author James Martin, PhD. “My first challenge was convincing the lab that it was true.” The ability of a compound to resist the development of bacterial resistance is also something of a double-edged sword. Typical antibiotics research involves finding a molecule that can kill bacteria, going through multiple generations of bacteria until they evolve resistance to it, looking at how exactly that resistance operates, and using that to reverse-engineer how the molecule works in the first place. But since SCH-79797 was proving irresistible, the researchers had nothing to reverse engineer from. “This was a real technical feat,” said Gitai. “No resistance is a plus from the usage side, but a challenge from the scientific side.”

This gave the research team the challenge of both trying to prove that bacteria wouldn’t develop resistance to SCH-79797, and figuring out how the compound works. To demonstrate that bacteria would not become resistant to the compound, Martin first tried a multitude of different assays and methods, none of which revealed the development of any resistance whatsoever. He then carried out serial passaging over 25 days, which involved exposing the bacteria to the drug over repeated generations. Bacteria take about 20 minutes per generation, so while this duration of serial passaging gave them millions of chances to evolve resistance, they still didn’t.

To check their methods, the scientists also serially passaged other antibiotics (novobiocin, trimethoprim, nisin, and gentamicin) and showed that bacteria quickly developed resistance to them. “… mutants that evolved increased resistance to antibiotics like trimethoprim and nisin did not demonstrate cross-resistance to SCH-79797,” the team noted. This apparent “irresistable” characteristic of SCH-79797 was the basis for naming derivative compounds, or Irresistins, that they subsequently developed. “This is really promising, which is why we call the compound’s derivatives ‘Irresistin,'” Gitai said.

The team also tested the compound against bacterial species that are known for their antibiotic resistance, including Neisseria gonorrhoeae, which is on the top five list of urgent threats published by the Center for Disease Control and Prevention. “Gonorrhea poses a huge problem with respect to multidrug resistance,” said Gitai. “We’ve run out of drugs for gonorrhea. With most common infections, the old-school generic drugs still work. When I got strep throat two years ago, I was given penicillin-G—the penicillin discovered in 1928! But for N. gonorrhoeae, the standard strains that are circulating on college campuses are super drug resistant. What used to be the last line of defense, the break-glass-in-case-of-emergency drug for Neisseria, is now the front-line standard of care, and there really is no break-glass backup anymore. That’s why this one is a particularly important and exciting one that we could cure.”

The researchers acquired from the World Health Organization repository a sample of the most resistant strain of N. gonorrhoeae, which is resistant to every known antibiotic, and “Joe showed that our guy still killed this strain,” Gitai said, referring to Joseph Sheehan, a co-first-author on the paper and the lab manager for the Gitai Lab. “We’re pretty excited about that.”

Without resistance to reverse engineer from, the researchers spent years trying to determine how the molecule kills bacteria, using a huge array of technologies, from classical approaches that have been around since the discovery of penicillin, through to cutting-edge technology. Martin called it the “everything but the kitchen sink” approach, and it eventually revealed that SCH-79797 uses two distinct mechanisms within one molecule.

The team likened this to using an arrow coated in poison. “The arrow has to be sharp to get the poison in, but the poison has to kill on its own, too,” said Benjamin Bratton, PhD, an associate research scholar in molecular biology and a lecturer in the Lewis Sigler Institute for Integrative Genomics, who is the other co-first-author.

The arrow targets the outer bacterial membrane, piercing through even the protective coat of Gram-negative bacteria, while the poison shreds folate, a fundamental building block of RNA and DNA. The researchers were surprised to discover that the two mechanisms operate synergistically, combining into more than a sum of their parts. “… the combination of two different antibacterial activities on the same molecular scaffold can, at least in the case of SCH-79797, produce a more potent antibacterial effect than co-treating with two antibiotics with the two separate targeting activities,” the investigators wrote. Bratton added, “If you just take those two halves—there are commercially available drugs that can attack either of those two pathways—and you just dump them into the same pot, that doesn’t kill as effectively as our molecule, which has them joined together on the same body.”

One problem with SCH-79797 was that it killed human cells and bacterial cells at roughly similar levels, so the researchers developed an SCH-79797 derivative, which they called Irresistin-16 (IRS-16), that they demonstrated was nearly 1,000 times more potent against bacteria than human cells, making it a promising antibiotic. As a final confirmation, the researchers demonstrated that they could use Irresistin-16 to cure mice infected with N. gonorrhoeae.

This poisoned arrow paradigm could revolutionize antibiotic development, said KC Huang, PhD, a professor of bioengineering and of microbiology and immunology at Stanford University who was not involved in the research. “The thing that can’t be overstated is that antibiotic research has stalled over a period of many decades,” Huang said. “It’s rare to find a scientific field which is so well studied and yet so in need of a jolt of new energy.” The synergy between two mechanisms of attacking bacteria, “can provide exactly that,” added Huang, who was previously a postdoctoral researcher at Princeton. “This compound is already so useful by itself, but also, people can start designing new compounds that are inspired by this. That’s what has made this work so exciting.”

Each of the two mechanisms targeted by the new compounds are present in both bacteria and in mammalian cells. Folate is vital to mammals, and both bacteria and mammalian cells have membranes. “This gives us a lot of hope, because there’s a whole class of targets that people have largely neglected because they thought, ‘Oh, I can’t target that, because then I would just kill the human as well,'” Gitai said.

“Here, we describe a promising compound, SCH-79797, and its derivative, IRS-16, that are effective in animals and address these key criteria with a unique dual-targeting MoA, the ability to kill both Gram-negative and Gram-positive pathogens, and an undetectably low frequency of resistance,” the authors concluded. “The acute need for new antibiotics to treat N. gonorrhoeae makes IRS-16 particularly promising small molecule candidate for future development … This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens … Thus, our findings identify and characterize a promising new antibiotic and provide a potential roadmap for future antibiotic discovery efforts.”

“A study like this says that we can go back and revisit what we thought were the limitations on our development of new antibiotics,” Huang further commented. “From a societal point of view, it’s fantastic to have new hope for the future.”

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