According to the UN Environment Programme, bacteria resistant to existing antibiotics cause approximately one million deaths each year — a toll expected to soar ten times higher by 2050 if researchers don’t develop alternative therapies. Some bacteria, including some strains of the gut pathogen Escherichia coli or the lung pathogen Klebsiella pneumoniae, are resistant to multiple antibiotics, limiting treatment options. These species belong to a category called ‘Gram-negative’, a historic classification that usefully groups a class of bacteria that use the same mechanism to make themselves hard to target, namely a cell wall structure that very actively pumps toxins back out of the bacterium. Members of this group possess two bacterial membranes, the outer of which is studded with fatty carbohydrates called lipopolysaccharides (LPS) that play an essential role in reinforcing the membrane’s integrity. Douglas Huseby, a microbiologist at Uppsala University in Sweden, and his colleagues are designing compound series that obstruct LPS synthesis as detailed in the paper, Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria. AstraZeneca previously developed a compound called AZ-1 that could inhibit this pathway but found that it underperformed. After screening other molecules with inhibitory potential, Huseby and his team found one with a similar structure to AZ-1. By merging the two molecules into one, the team developed a potent blocker. X-ray crystallography experiments at Diamond’s I04-1 beamline revealed that the new compound inhibits an essential enzyme by binding to part of the active site. After tweaking the molecule to improve its properties, the team repeated their crystallography work to confirm it bound to the same site on the enzyme. Preclinical tests in mice show a single dose of the compound is safe and effective, underscoring the molecule’s encouraging potential.
No antibiotic is invulnerable to bacterial resistance. Regardless of what bacterial components the drugs target, bacteria evolve defences against every class of the drugs. Now more than ever, clinicians need novel antibiotics that target new microbial features, slowing down the emergence of resistant, untreatable superbugs, but new antibiotics have not completed the journey from the lab to the clinic since the 1970s.
During the mid-20th century, scientists turned to nature to discover antibiotics, but that has become untenable today. “Each new antibiotic you find is about ten times harder to find than the previous one,” Huseby said. Today, scientists focus more attention on designing new antibiotics instead.
The enzymes that synthesize the fatty sugar lipopolysaccharide (LPS) present an alluring, fresh target. LPS is found on the outer surface of Gram-negative bacteria, which have a thin cell wall and two cell membranes. Several Gram-negative bacteria cause severe infections in humans and have evolved resistance to multiple antibiotics. These include Escherichia coli, a common cause of diarrhoea, and Klebsiella pneumoniae, a tough-to-treat superbug behind pneumonia and meningitis.
LPS is essential in Gram-negative bacteria, as it reinforces the structural integrity of Gram-negative bacteria's outer membrane, as noted in Mechanism of outer membrane destabilization by global reduction of protein content. Owing to its importance, enzymes involved in its synthesis make good targets for drug design. What's more, humans lack this synthesis pathway, meaning drugs could theoretically kill bacteria without having collateral effects by disrupting human proteins.
LPS anchors to the outer surface of the bacteria using a fatty molecule called lipid A, and the enzyme that synthesizes this anchor, LpxH, is present in 70% of Gram-negative bacteria, so Huseby and his colleagues wanted to design an antibiotic that could inhibit this enzyme. Since this enzyme is conserved in many species, drugs that target it could potentially have broad-spectrum use, much like the cephalosporin drugs used to treat many Gram-negative microbes. (New agents for the treatment of infections with Gram-negative bacteria: restoring the miracle or false dawn?)
AstraZeneca already developed a drug candidate that inhibits LpxH called AZ1; however, the compound was only effective in bacteria if the researchers turned off their efflux pumps — portals on the surface that jettison antibiotics (Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay). “It’s not uncommon that you aren’t able to overcome the efflux problem,” Huseby said.
The team decided to search for compounds with inhibitory potential. After screening multiple hits, they landed on a molecule called JEDI-852. This candidate caught their attention because some bacteria in their screen evolved resistance to it by mutating their LpxH gene, suggesting this compound targets the enzyme.
Unexpectedly, JEDI-852 and AZ1 showed striking similarities. By observing the molecules side by side, Huseby’s team noticed that they share a common core.They decided to synthesize a merged version, with unique functional groups from AZ1 at one end and ones from JEDI-852 at the other. Their newly fashioned molecule, which they called JEDI-1444, proved superior at inhibiting LpxH than AZ1. It even worked in bacteria with functioning efflux pumps, suggesting it is potent enough to kill bacteria even if they expel some of the compound from the cell.
Next, they solved the crystal structure of JEDI-1444 bound to LpxH from E. coli and K. pneumoniae. They discovered that the compound partially occupies the same cavity as the substrates for lipid A, suggesting the molecule might work by obstructing the enzyme's active site, though further experiments are needed to confirm this mechanism of action.
Although JEDI-1444 proved itself a potent inhibitor, some of its properties were undesirable: enzymes in mice and humans break down the compound, making it chemically unstable; it doesn't dissolve well in water; and serum proteins in blood can block its antibiotic activity, potentially by sequestering the molecule away from bacteria. Huseby’s team had to modify the compound’s structure. That involved turning to the crystal structure to find regions where they could make modifications without preventing the compound from docking at the active site, which revealed they should avoid making edits to the core that was common to the initial two compounds. To tweak the compound without disturbing the core, they made changes to both ends of the molecule. They ended up with two new molecules called EBL-3647 and EBL-3599 bearing corrections that improved the stability and solubility of the compounds and reduced their interactions with serum proteins. Further X-ray crystallography experiments revealed that these compounds bind to the same region on the LpxH as JEDI-1444, confirming that they inhibit the enzyme in a similar fashion
Finally, they administered both compounds to mice and found that neither induced side effects, suggesting these compound series might be safe. Testing them out on mice infected with K. pneumoniae showed that a single dose of either compound could partially lower bacterial numbers. Infections with E. coli were more promising: the same treatment cleared the infections to undetectable levels.
In the next steps, researchers will need to continue assessing the compounds’ safety and efficacy with further animal tests and subsequent human trials. This will lead to the development of the final drug over time. The molecules have favourable qualities so far, but Huseby cautioned that clinical studies are where problems often first show up. Referring to past research, he said, “once we expand to even larger groups of people, suddenly, we see toxicity that we didn’t see in smaller clinical trials.”
The chances that a new antibiotic candidate will break through to pharmacy are generally slim. However, so are our odds of outpacing the growing threat of antibiotic resistance if researchers don’t jump on this high-risk venture. It’s time for funders to recognize that the lives saved by occasional successes will far outweigh the risks taken. For all we know, JEDI-1444’s derivatives could be the much-needed additions to every doctor’s medicine cupboard.
To find out more about I04-1 or discuss potential applications, please contact principal beamline scientist Frank von Delft: [email protected].
As the threat of antibiotic resistance looms, scientists are designing new drugs to fight off bacteria. A team of microbiologists developed a potent inhibitor against E. coli and other Gram-negative bacteria. X-ray crystallography experiments unveiled how the compound latches onto an enzyme needed for the synthesis of a fatty sugar on the outer-membranes of these bacteria, presenting a new compound-enzyme interaction with therapeutic potential.
The research presented in this paper was conducted as part of the ND4BB ENABLE Consortium and has received support from the Innovative Medicines Initiative Joint Undertaking under grant no. 115583, resources of which are comprised of financial contributions from the European Union’s seventh framework program (FP7/2007-2013) and EFPIA companies’ in-kind contribution. The views expressed in this article are the views of the authors, and neither IMI nor the European Union or EFPIA is responsible for any use that may be made of the information contained herein. We acknowledge the support of ENABLE-2, a national platform for antibiotics development funded by the Swedish Research Council and the Swedish National Research Programme on Antibiotic Resistance (Dnr 2021-06603).
Huseby, DL et al. Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria. PNAS 121(15), e2317274121. DOI: 10.1073/pnas.2317274121
Image credits: this article (10.1073/pnas.2317274121) under Creative Commons Attribution License 4.0 (CC BY)
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