Researchers at the University of California, San Francisco (UCSF), have reported on an approach to tackling antibiotic resistance that effectively redesigns existing antibiotic molecules so that they can evade bacterial resistance mechanisms. By devising a set of molecular LEGO pieces that can be altered and joined together to form larger molecules, the researchers created what they hope is the first of many ‘rebuilds’ of drugs that had been shelved due to antibiotic resistance.
In a paper in Nature, the team describes application of the technology to design new streptogramin-type antibiotics. “The aim is to revive classes of drugs that haven’t been able to achieve their full potential, especially those already shown to be safe in humans,” said lead author Ian Seiple, PhD, an assistant professor in the UCSF School of Pharmacy’s Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute (CVRI). “If we can do that, it eliminates the need to continually come up with new classes of drugs that can outdo resistant bacteria. Redesigning existing drugs could be a vital tool in this effort.”
Seiple, together with James Fraser, PhD, a professor in the School of Pharmacy’s Department of Bioengineering and Therapeutic Sciences in the UCSF School of Pharmacy, and colleagues, report on their developments in a paper titled, “Synthetic group A streptogramin antibiotics that overcome Vat resistance.”
Antibiotic resistance is one of the world’s most urgent public health threats. In the U.S. alone, tens of thousands of deaths result each year from drug-resistant strains of common bacteria such as Staphylococcus aureus and Enterococcus faecium, which can cause virtually untreatable hospital-acquired infections. Perilously few new classes of antibiotics are being developed to fight infections that have become resistant to traditional treatments, and bringing any new drugs to market could take decades.
Streptogramins, like most other antibiotics, are derived from naturally occurring antibiotic compounds produced by other organisms (usually bacteria) that are then tweaked to optimize their performance in the human body. But bacteria can develop mechanisms to resist their effectiveness, the authors explained. “Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics.”
Streptogramins disable bacteria by effectively gumming up the works in bacterial ribosome, making it impossible for the bacteria to make proteins. Streptogramins had been very effective against S. aureus infections, until the bacteria evolved a resistance mechanism. Bacteria that are resistant to streptogramins produce proteins called virginiamycin acetyltransferases (Vats), which recognize these antibiotics when they enter the bacterial cell.
The Vats grab the drug and chemically deactivate it before it can bind to the ribosome, rendering it useless. “Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome,” the authors wrote.
Seiple figured that there must be a way to make further changes to the streptogramin drug molecule that would allow it to evade capture by the Vat proteins. The team set out to build new streptogramins from the ground up, rather than modifying existing structures. To make the building process easier, Qi Li, PhD, a postdoctoral fellow in the Seiple lab and co-first author on the paper, created seven molecular modules that can be tweaked as needed to build a set of variations on the streptogramin molecule.
“This system allows us to manipulate the building blocks in ways that wouldn’t be possible in nature,” said Seiple. “It gives us an efficient route to re-engineering these molecules from scratch, and we have a lot more latitude to be creative with how we modify the structures.” Once Seiple and Li had their building blocks, the next step was to get a molecular-level view of the chemistry involved in order to better understand how to modify and piece together those molecular LEGOs. To do that, Seiple teamed up with Fraser, who specializes in creating visual models of biological molecules.
Fraser stated, “My lab’s contribution was to say, ‘Now that you’ve got the seven pieces, which one of them should we modify and in what way?'” To answer that question, Jenna Pellegrino, a graduate student in the Fraser Group and co-first author on the paper, used two complementary techniques, cryo-electron microscopy and x-ray crystallography, to create three-dimensional pictures of the drug at near-atomic resolution, as well as its target, the bacterial ribosome, and the Vat protein that thwarts antibiotic activity. Using the models, Li, Pellegrino, Seiple, and Fraser could see which parts of the streptogramin molecule are essential to antibiotic function. Then, Li was free to modify the drug’s non-essential regions, to find alterations that prevented Vats from interacting with the drug, while still allowing it to bind to its ribosomal targets and disable the bacterium.
The team found that two of the seven building blocks seemed to offer potentially interesting sites for modification. They made variations of the drug that contained tweaks in those regions and found that the variants were active in dozens of strains of pathogenic bacteria. The researchers also tested their most promising candidate against streptogramin-resistant S. aureus infections in mice, and found that the compound was was greater than 10 times more effective than other streptogramin antibiotics. “One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection,” the authors noted.
“By combining modular chemical synthesis, antibacterial evaluation, in vitro analysis, and high-resolution cryo-electron microscopy, we have developed a pipeline for the synthesis and optimization of group A streptogramin antibiotics,” they stated. “Our approach enabled the preparation of novel analogues by means of building block variation and late-stage diversification, providing valuable structure–activity relationships for the class.”
Seiple points out that knowledge gained through the collaborative experiments could be applied to modifying many other antibiotics. “We learned about mechanisms that other classes of antibiotics use to bind to the same target,” he said. “In addition, we established a workflow for using chemistry to overcome resistance to antibiotics that haven’t reached their potential.” As the investigators concluded in their paper, “Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.”
Seiple aims to continue refining these synthetic streptogramins, and hopes that the reengineered antibiotics could ultimately be further developed and tested in human trials. He and Fraser plan to continue working together on reviving other antibiotics that have been shelved because of microbial resistance, refining a set of tools that can help researchers stay one step ahead of bacterial evolution. “It’s a never-ending arms race with bacteria,” said Fraser. “But by studying the structures involved—before resistance arises—we can get an idea of what the potential resistance mechanisms will be. That insight will be a guide to making antibiotics that bacteria can’t resist.”