Crystallography reveals how an E. coli drug candidate inhibits an essential enzyme
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’ that do not retain a violet ‘Gram’ dye very well when stained and viewed under the microscope. 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 drug candidates that obstruct LPS synthesis. 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 drug 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 drug is safe and effective, underscoring the drug’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.
Attacking bacteria from the outside
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?)
Merging medicines
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 drug from the cell.
Read more on Diamond website
Image: X-ray crystallography experiments at Diamond’s I04-1 beamline revealed that the new drug, JEDI-1444, binds to the LPS-synthesizing enzyme in the same place as substrates, suggesting it may interfere with the active site. Image credit: Douglas Huseby; adapted from the PNAS publication in accordance with CC-BY 4.0 license.
