New routes for antibiotic development against P. aeruginosa

The creation of a new antibiotic from discovery to clinical use can take 10-15 years (1). This already lengthy process is riddled with checks and balances that only let 27 percent of new drugs enter clinical trials, and only 8-16 percent of those make it onto local pharmacy shelves (2). Developing new antibiotics for pathogens that are resistant poses another challenge as researchers must avoid using the same tactics of already known ineffective antibiotics.

Pseudomonas aeruginosa, one of the multi-drug resistant ESKAPE pathogens (named for the top virulent and antibiotic resistant pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp), tops the World Health Organization’s list of pathogens most in need of new antibiotics (3). It accounts for 32,000 infections a year in the USA alone (4). Now, researchers have identified novel routes for effective antibiotic discovery against P. aeruginosa and laid out a guide for others seeking to make effective antibiotics against this pathogen (5).

A woman with red curly hair wears a lab coat and uses a pipet tip to pick up a bacterial colony from an agar plate in a research laboratory.

Coauthor Morgan Gugger from Hergenrother’s team selects a single colony of bacteria from an agar plate.

Credit: Paul Hergenrother

“A big goal of our work is to develop novel antibiotics for gram-negative pathogens, specifically the ones that are very hard to treat like the ESKAPE pathogens,” said coauthor Paul Hergenrother, a chemist at the University of Illinois. “There hasn't been a new class of FDA-approved antibiotics in over 50 years, so we've been thinking about that for a decade and working on various ways.”

What makes P. aeruginosa a difficult egg to crack has to do with the difficulty in maintaining antibiotics once they enter the cell. This bacterium is equipped with a specialized outer membrane that allows few things to get inside as well as many efflux pumps that make quick work of removing any unwanted antibiotics. For antibiotics to work, they must reach a critical threshold within the bacterium, and without this accumulation, the drugs quickly become ineffective.

Hergenrother’s team sought to identify what kinds of chemicals could accumulate within P. aeruginosa and perhaps find similar characteristics amongst the chemicals that are key to accumulating in this pathogen.

“If one were to only focus on antibacterial activity, that limits the number of chemical compounds dramatically to ones that will kill the bacteria,” said Hergenrother.

Using a mix of commercially available chemicals, naturally-derived chemicals, and newly synthesized ones created by the team, the researchers tested if these compounds entered the cell, and more importantly, if they accumulated. They identified the accumulating compounds using a technique that can sort molecules based on size, polarity, charge, and other distinguishing features. Once they discovered which chemicals make good accumulators, they input this data into a machine-learning algorithm to identify common features among these chemicals. They found that molecules with positive surface charges and readily available hydrogen bond donors were better at accumulating in the cell. Specifically having diamines, guanidiniums, and pyridiniums boosted the accumulation rates of these compounds.

A big goal of our work is to develop novel antibiotics for gram-negative pathogens, specifically the ones that are very hard to treat, like the ESKAPE pathogens.
- Paul Hergenrother, University of Illinois

“The problem is so important and very difficult, and this is a major advancement,” said Dirk Bumann, a biochemist from the University of Basel, who was not involved in this study. Bumann noted that diamines typically have a toxicity problem in in vivo studies. “They are long known to improve the efficacy of antibiotics, but they come with very serious safety liabilities. But this is a great paper with many open questions to follow up on.”

The team identified that these successful chemicals enter the cell in a porin-independent manner, meaning that they do not rely on P. aeruginosa’s common import machinery . This result was shocking when compared to E. coli, which uses a general porin for nearly all antibiotic imports.

Wanting to put their guidelines to the test, the researchers chemically modified fusidic acid (FA), an antibiotic that is effective against gram-positive but not gram-negative bacteria. Hoping to enhance its accumulation abilities and subsequent effectiveness in killing P. aeruginosa, they added a positively charged polyamine linker to the compound. Once inside the gram-negative bacteria, a hydrolysis reaction cleaved the molecule and released FA inside the cell. Comparing the chemically altered FA to the general FA, the modified drug had a 64 to 256 fold increase in activity and accumulated 20 times more in the cell.

“What we're trying to do is to develop guidelines that researchers can use to take an antibiotic that say they discovered through a biochemical screen or whatever, and then by using these guidelines, engineer the chemical traits that enable the compound now to get in and kill the bacteria,” said Hergenrother. “Because we made all these compounds, we can systematically alter them and test those hypotheses experimentally.”