Tag: antibiotics

Tearing down bacterial membranes with new antibiotics

Tearing down bacterial membranes with new antibiotics

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.

Discovery paves way for next-generation medications

Discovery paves way for next-generation medications

As the problem of antibiotic resistance continues to grow, we need new drugs that the bad bacteria in our bodies don’t already know how to avoid. New research by scientists at McGill University represents a major step forward in our ability to develop medicines whose effectiveness will endure in the battle against infections.

The study, published in the prestigious journal Nature, has revealed how molecular machinery inside nature’s microbes builds antibiotics. Researchers have been working on this problem for decades, and this new insight represents a major step forward in our ability to create new drugs and medicines.

Scientists Angelos Pistofidis and Martin Schmeing used the Canadian Light Source (CLS) at the University of Saskatchewan to take groundbreaking pictures of the molecular machinery’s crystal structure.

The molecular machines that Pistofidis and Schmeing studied are called nonribosomal peptide synthetases, or NRPSs. They build some of the most important compounds in current health care and environmental treatments, including antibiotics, anti-cancer agents, and immunosuppressants.

“They have an immense number of applications,” says Pistofidis. “For example, the peptide cyclosporin has been used many, many times as an immunosuppressant for organ transplant operations.”

The breakthrough in their project was capturing images of the NRPS during a key step in the process of building antibiotics. Previously, they had identified the steps involved in NRPS’s production process, but the details were hazy. The synchrotron played a key role in their work.

“The CLS is a world-class establishment. You can very rapidly and very efficiently collect data. It made the whole experience of collecting data on a very complex crystal, like the one that we presented in the paper, quite efficient,” says Schmeing.

Getting the NRPS machine to pause at this step took Pistofidis four years of work, while Schmeing has been working on uncovering the details of this whole process for 15 years.

Read more on CLS website

Looking into the heart of an antibiotic killer

Looking into the heart of an antibiotic killer

β-lactam-based antibiotics currently account for about 65% of all applied antibiotics, due to their broad-spectrum of activity and favorable safety profile, making this class of drugs the most common clinical approach for treating bacterial infections. Examples of these drugs, which contain a β-lactam ring in their structure, include naturally occurring penicillins, and synthetic cephalosporins, monobactams, and carbapenems. Antibiotics with a β-lactam core target bacterial transpeptidases—enzymes necessary for cell-wall synthesis—and they block the formation of cross-bridges between adjacent peptidoglycan chains, leading to bacterial death. Overuse of β-lactam antibiotics has led to an increase in microorganisms with multidrug resistance. In β-lactam antibiotics, this resistance is driven primarily by bacterial enzymes called b-lactamases. Researchers have now revealed the crystal structure, binding, and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from the emerging pathogen Stenotrophomonas maltophilia using the U.S. Department of Energy’s Advanced Photon Source (APS). Drug discovery based on the details captured in this study could contribute key information to counteract antimicrobial resistance and provide tools in future pandemics. The results were published in the journal Nature Communications.

Read more on the APS website

Image: Fig. 1. TR-SSX crystal structure of moxalactam of the active site of L1 MBL, L1 active site structure at 150 ms with hydrolyzed moxalactam (in yellow-red-blue), zinc (magenta) and protein residues (in silver-blue-red).

Lightsources.org virtual symposium recording

Lightsources.org virtual symposium recording

Lightsources.org was delighted to welcome over 500 attendees to our live virtual symposium to mark the 75th Anniversary of the first direct observation of synchrotron light in a laboratory. The event, which was chaired by Sandra Ribeiro, Chair of lightsources.org and Communications Advisor for the Canadian Light Source, was held on the 28th April 2022 and you can watch the recording via the YouTube link below.

We received some lovely feedback after the live event, including this comment from Jeffrey T Collins at the Advanced Photon Source, Argonne National Laboratory in Illinois.

 “I have worked at the Advanced Photon Source for over 32 years and I learned many things during this event that I never knew before.  It was quite informative.  I look forward to re-watching the entire event.”

Jeffrey T Collins, Mechanical Engineering & Design Group Leader at Argonne National Laboratory

The symposium began with a historical introduction from Roland Pease, freelance science broadcaster who has been an enthusiastic support of light sources for many years.

Roland’s talk was followed by experts from the field giving talks on their perspectives of synchrotron light related achievements that have been made since the 1st laboratory observation on the 24th April 1947.

Speakers were:

• Nobel Laureate Prof. Ada Yonath (Weizmann Institute of Science)

• Prof. Sir Richard Catlow (University College London)

• Prof. Henry Chapman (DESY)

• Dr Paul Tafforeau (ESRF)

• Dr Gihan Kamel (SESAME and member of the AfLS Executive Committee).

There followed a panel discussion with special guests who all made huge contributions to the development of the field. Our special guests were:

Herman Winick – Prof. of Applied Physics (Research) Emeritus at SLAC)

Ian Munro – Initiator of synchrotron radiation research at Daresbury Laboratory ,Warrington UK in 1970

Giorgio Margaritondo – one of the pioneers in the use of synchrotron radiation and free electron lasers

Gerd Materlik – former CEO of Diamond Light Source, the UK’s synchrotron science facility

Lightsources.org is hugely grateful to all the speakers, special guests and attendees who contributed to this event and made it such a special anniversary celebration for the light source community.

If you have any feedback or memories to share, please do contact Silvana Westbury, Project Manager, at webmaster@lightsources.org

For news, jobs, events and proposal deadlines, please visit the homepage

“foot-2-foot” interaction sheds light on bacterial conjugation

“foot-2-foot” interaction sheds light on bacterial conjugation

Bacteria possess mechanisms to establish communication between cells. This is especially important in bacterial conjugation, a process that allows bacteria to share genetic material. This is often used by bacteria to transfer antibiotic resistance genes and other virulence factors to neighbor cells, increasing the antibiotic resistance spread.

Now, a research team of ALBA scientists report the structural mechanism by which two proteins, Rap and Rco, act together to regulate conjugation. Rco is a repressor of conjugation, whereas Rap binds Rco and prevents Rco-mediated conjugation repression, thus resulting in an activation of the conjugation mechanism. The main results of the study show that Rap contains a binding pocket were a short peptide can bind, producing structural changes in Rap that forces its tetramerization, releasing Rco for blocking conjugation. Tetramerization occurs through an interaction that scientists named “foot-2-foot”, which differs significantly from the model proposed for other proteins of the Rap family.

Read more on the ALBA website

Image: RappLS20 tetramerization, side view of the peptide-bound tetramer. The red arrows indicate the loops connecting helices H4 and H5. (C) Zoom of the area around the N-terminus of helix H4, showing the insertion of this helix into the opposite monomer. The homotetramerization caused by the foot-2-foot interactions of the NTDs of RappLS20 provides an explanation for the activation of the RcopLS20 partner. In the absence of the peptide, the NTDs are positioned such that they allow the interaction with RcopLS20. However, upon binding the signaling peptide, the NTDs shift outwards, facilitating the formation of the homotetramer, leading to a change of the interaction surface of the NTDs that is no longer available for interactions with RcopLS20

Helping our immune systems bypass antibiotic resistance

Helping our immune systems bypass antibiotic resistance

Over 700,000 people die each year due to drug-resistant diseases and this figure could increase to 10 million per year by 2050, according to a 2019 report.

As the search continues for new antibiotics to treat drug-resistant infections, a group of researchers used the Canadian Light Source (CLS) at the University of Saskatchewan to address the problem from a different direction, by trying to weaken the ability of bacteria to develop resistance in the first place.

“The goal is to knock the bacterial cells down in terms of their resistance,” said Dr. Anthony Clarke, Professor and Dean of Science at Wilfrid Laurier University and adjunct professor at the University of Guelph. “We haven’t been successful over the last 30 years in finding new classes of antibiotics so, in the short term, we’re trying to weaken the cells so our own immune system can take over to fight infection.”

The target for his team’s work is peptidoglycan, which gives bacterial cell walls their rigidity. “Think of it as building a brick wall around the bacteria’s cells,” said Clarke. Since peptidoglycan can be broken down by lysozyme, an enzyme that exists in human immune systems, bacteria have developed strategies that block these enzymes by modifying their peptidoglycan, thereby “cementing the bricks in place,” and resisting our defences.

Read more on the Canadian Light Source website

Image: Dr. Clarke inspecting flasks of bacterial cultures in a student laboratory.

Using state-of-the-art nanocarriers to beat bacterial resistance

Using state-of-the-art nanocarriers to beat bacterial resistance

Novel stabiliser-free cubosomes can transport antimicrobial peptides and promote wound healing

In 2018, in England alone, there were an estimated 61,000 antibiotic resistant infections – a 9% rise on the previous year. Infections that don’t respond to antibiotics have the potential to cause bloodstream infections and may require patients to be admitted to hospital. The numbers of antibiotic-resistant bloodstream infections rose by a third between 2014 and 2018. The rise in antibiotic-resistant bacteria is a growing concern worldwide, prompting a search for new antibiotics and alternative strategies for fighting bacteria. One promising approach is the design of lipid-based antimicrobial nanocarriers. However, most of the polymer-stabilised nanocarriers are cytotoxic. In work recently published in  Advanced Functional Materials,  a team of Swiss researchers designed a novel, stabiliser-free nanocarrier for the antimicrobial peptide LL-37 that also promotes wound healing. They demonstrated that stabiliser-free cubosomes show promise as advanced cytocompatible nanovehicles for nutrient and drug delivery. 

Vertebrates have two main immune strategies. In simple terms, the adaptive (or acquired) immune system responds to specific pathogens by producing antibodies. The innate immune system is older (in evolutionary terms) and is found in all kinds of life, from plants and fungi to insects and multicellular organisms. The innate immune system makes use of less specific defence mechanisms, including physical barriers (such as skin or bark), clotting factors in blood or sap, and specialised cells that attack foreign substances. 

Read more on the Diamond Light Source website

Image : Graphical representation of a cubosome. The coloured surface resembles the lipid-water interface with the confined water channels. The channel diameter is typically in the range of 10 nanometres, with the overall size of the cubosomes being several hundred nanometres.

Visualising the bionanomachines that create potent antibiotics

Visualising the bionanomachines that create potent antibiotics

… and other modern drugs.

Researchers from McGill University and Yale University used the Canadian Light Source (CLS) at the University of Saskatchewan to make a discovery that could help design future therapeutic drugs. The research team studied how mega-enzymes, known as nonribosomal peptide synthetases (NRPSs), create potent antibiotics, immunosuppressants and other modern drugs.

In a paper featured on the cover of the May 2020 issue of Nature Chemical Biology, the team reports how they were able to visualize an NRPSs’ mechanical system using the CMCF beamline at the CLS.

>Read more on the Canadian Light Source website

Image: Associate Professor Schmeing in the lab

Study offers new target for antibiotic resistant bacteria

Study offers new target for antibiotic resistant bacteria

As antibiotic resistance rises, the search for new antibiotic strategies has become imperative. In 2013, the Centers for Disease Control estimated that antibiotic resistant bacteria cause at least 2 million infections and 23,000 deaths a year in the U.S.; a recent report raised the likely mortality rate to 162,044.
New Cornell research on an enzyme in bacteria essential to making DNA offers a new pathway for targeting pathogens. In “Convergent Allostery in Ribonucleotide Reductase,” published June 14 in Nature Communications, researchers used the MacCHESS research stations at the Cornell High Energy Synchrotron Source (CHESS) to reveal an unexpected mechanism of activation and inactivation in the protein ribonucleotide reductase (RNR).

Understanding the “switch” that turns RNR off provides a possible means to shut off the reproduction of harmful bacteria.
RNRs take ribonucleotides, the building blocks of RNA, and convert them to deoxyribonucleotides, the building blocks of DNA. In all organisms, the regulation of RNRs involves complex mechanisms, and for good reason: These mechanisms prevent errors and dangerous mutations.

>Read more on the CHESS website

Image: William Thomas, a graduate student in the field of chemistry and chemical biology, collects data on ribonucleotide reductase.

How stained glass can help in the battle against superbugs

How stained glass can help in the battle against superbugs

Ancient skills meet cutting edge technology in the battle against antibiotic resistance

Bacteria can form colonies (known as biofilms) on the surface of objects. This is a particular problem when it occurs on medical devices implanted into the body, such as catheters, prosthetic cardiac valves and intrauterine devices, as biofilms can display resistance to both antibiotics and the body’s immune response. Any incision into the body risks a surgical infection, and if a biofilm takes hold it can be difficult to eradicate. With the rise in antibiotic resistance, scientists are seeking new ways to prevent infections, and there is increasing interest in impregnate medical devices with antimicrobial substances. In work recently published in ACS Biomaterials Science & Engineering, researchers from Aston University in Birmingham, led by Dr Richard Martin, explored the antimicrobial potential of phosphate glasses doped with cobalt, and found them to be effective against Escherichia coli, Staphylococcus aureus and Candida albicans when placed in direct contact, suggesting that cobalt-doped bioactive glasses could be developed with antimicrobial properties. The technique they discovered is similar to those used to make stained glass in medieval times.

>Read more on the Diamond Light Source website
Image: Images of the copper (left) and cobalt (right) doped bioactive glasses.
Credit: Dr Richard Martin
First experiments reveal unknown structure of antibiotics killer

First experiments reveal unknown structure of antibiotics killer

DESY-led international collaboration obtains first scientific results from European XFEL

An international collaboration led by DESY and consisting of over 120 researchers has announced the results of the first scientific experiments at Europe’s new X-ray laser European XFEL. The pioneering work not only demonstrates that the new research facility can speed up experiments by more than an order of magnitude, it also reveals a previously unknown structure of an enzyme responsible for antibiotics resistance. “The groundbreaking work of the first team to use the European XFEL has paved the way for all users of the facility who greatly benefit from these pioneering experiments,” emphasises European XFEL managing director Robert Feidenhans’l. “We are very pleased – these results show that the facility works even better than we had expected and is ready to deliver new scientific breakthroughs.” The scientists present their results, including the first new protein structure solved at the European XFEL, in the journal Nature Communications.

“Being at a totally new class of facility we had to master many challenges that nobody had tackled before,” says DESY scientist Anton Barty from the Center for Free-Electron Laser Science (CFEL), who led the team of about 125 researchers involved in the first experiments that were open to the whole scientific community. “I compare it to the maiden flight of a novel aircraft: All calculations and assembly completed, everything says it will work, but not until you try it do you know whether it actually flies.”

The 3.4 kilometres long European XFEL is designed to deliver X-ray flashes every 0.000 000 220 seconds (220 nanoseconds). To unravel the three-dimensional structure of a biomolecule, such as an enzyme, the pulses are used to obtain flash X-ray exposures of tiny crystals grown from that biomolecule. Each exposure gives rise to a characteristic diffraction pattern on the detector. If enough such patterns are recorded from all sides of a crystal, the spatial structure of the biomolecule can be calculated. The structure of a biomolecule can reveal much about how it works.

>Read more on the DESY website and on the European XFEL website

Image: Artist’s impression of the experiment: When the ultra-bright X-ray flashes (violet) hit the enzyme crystals in the water jet (blue), the recorded diffraction data allow to reconstruct the spatial structure of the enzyme (right).
Credit: DESY/Lucid Berlin

ALS passes the 7000-protein milestone

ALS passes the 7000-protein milestone

The eight structural biology beamlines at the ALS have now collectively deposited over 7000 proteins into the Protein Data Bank (PDB), a worldwide, open-access repository of protein structures. The 7000th ALS protein structure (entry no. 6C7C) is an enzyme from Mycobacterium ulcerans (strain Agy99), solved with data from Beamline 5.0.2. This bacterium produces a toxin that eats away at skin tissue, causing what’s known as Buruli ulcers (Google at your own risk!). The bacterium is antibiotic-resistant, and treatment involves the surgical removal of infected tissues, including amputation.

The enzyme structure was solved by a group from the Seattle Structural Genomics Center for Infectious Disease (SSGID), whose mission is to obtain crystal structures of potential drug targets on the priority pathogen list of the National Institute of Allergy and Infectious Diseases (NIAID). As of May 2018, SSGCID has deposited 1090 structures in the PDB, with data for more than a quarter of those collected at ALS beamlines.

>Read more on the Advanced Light Source website

Image: PDB 6C7C: Enoyl-CoA hydratase, an enzyme from M. ulcerans (strain Agy99).

How dolphins could potentially lead to new antibiotics

How dolphins could potentially lead to new antibiotics

The world is currently living through a multidrug resistance problem, where antibiotics that traditionally work are not effective anymore. A European team of scientists at the University of Hamburg (Germany), University of Munich (Germany), University of Bordeaux (France), University of Trieste (Italy) and University of London (UK) have studied how some peptides in dolphins target bacterial ribosomes and hence, could provide clues about potential new antibiotics.

Proline-rich antimicrobial peptides (PrAMPs) are antibacterial components of the immune systems of animals such as honey bees, cows and, as this study proves, bottlenose dolphins. These peptides are a first response for the killing of bacteria. In humans, antimicrobial peptides (AMPs) mainly kill bacteria by disrupting the bacterial cell membrane, but so far no evidence of PrAMPs has been found. PrAMPs have a different mechanism of action to AMPs: they pass through the membrane of the cell without perturbing it and bind to ribosomes to inhibit protein synthesis.

The European team have been studying the mechanism of action of bacteria killing peptides in animals: “We want to compare PrAMPs from different organisms to mechanistically understand how these peptides inhibit bacteria”, Daniel Wilson explains.

>Read more on the European Synchrotron website

Illustration showing the mechanism of Tur1A. (entire image: here)
Credits: D. Wilson