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Being able to produce a 3D molecular image of a drug as it binds to its target protein allows detailed analysis of the underlying molecular mechanisms. This, in turn, provides opportunities how these drugs could be modified to improve antibacterial activity and to overcome drug resistance.
β-lactam antibiotics are the most widely used class of antibacterial agents, so-called because they contain a particular chemical compound (a ring of four atoms called β-lactam) in their molecular structure. This structure interacts with bacteria and prevents them from constructing cell walls and destroys them. However, bacteria are becoming resistant to a number of these drugs, particularly through a process called β-lactamase-catalysed hydrolysis. β-lactamases are enzymes produced by the bacteria that break the β-lactam ring open by a chemical reaction called hydrolysis and thus deactivate the antibiotics’ properties.
Taniborbactam (also known as VNRX-5133) is a member of a new antibacterial drug class (bicyclic boronates) currently in clinical development that inhibits β-lactamase. A partnership of UK universities conducted macromolecular crystallography on beamlines I04 and I24 at Diamond to show how the drug binds with, and inhibits a broad range of β-lactamases. These ongoing studies are playing a vital role in the development of a potent new broad spectrum β-lactamase inhibitor that will aid the fight against antibiotic resistance.
Another UK study has also been investigating the bicyclic boronate drug class which are broad spectrum inhibitors of the serine- and metallo- β-lactamase families. The study used biophysical methods, including crystallographic analysis of a bicyclic boronate in complex with AmpC enzymes, at Diamond on beamline I04, to investigate the binding mode of these molecules to clinically important β-lactamases which are responsible for the antibacterial resistance seen in this drug class.
Burkholderia pseudomallei is the bacterium that causes the tropical disease called meliodosis (or Whitmore’s disease). The disease is responsible for a range of signs and symptoms that include mild fever, pneumonia, abscesses, and brain inflammation that can lead to death. Infection is normally transmitted through polluted water and improper treatment can lead to a death rate above 40%. The bacterium has a high resistance to antibiotics and an ability to overcome a host’s immune system which results in a high occurrence of latent infections, where people show symptoms of the disease many years after initial infection.
Researchers from the University of Leicester set out to understand the molecular mechanisms and protein structures of B. pseudomallei to provide a platform for the development of new drugs to counter the disease. Crystal structures were developed on the I03 beamline at Diamond which uncovered the role of specific enzymes that play an important role in the functioning of the bacterium and its susceptibility to different antimicrobial agents. Further research will continue on promising drug targets to prevent bacterial infection and prevent reactivation of old infections.
Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop new antibiotics with improved efficacy.
A research group from the University of Cambridge and the US National Institutes of Health combined to use a fragment-based approach to design inhibitors to a promising drug target called tRNA methyltransferase. This approach screens many small chemical compounds that may bind weakly to a biological target and then developing into potential drugs that have a much stronger affinity. X-ray data sets were collected on beamlines I02, I03, I04-1, I04 and I24 at Diamond and identified several compounds that show promising activity against mycobacterial species to overcome drug resistance and these are currently undergoing further investigation.
The collaborative European ENABLE project between industry, academia and biotech organisations has been established to combat antibiotic resistance. In particular the project is working to advance the development of potential antibiotics against multi-drug resistant Gram-negative infections. Imidazopyrazinones are a new class of antibacterial agents that target bacterial topoisomerases which are enzymes that assist the winding of DNA. A new pan-European study has solved the crystal structure of a number of these compounds which provides valuable insights into how these drugs work and how they could be used in the future. The study included macromolecular crystallography research performed on the I03 beamline at Diamond.
Pseudomonas aeruginosa is a Gram-negative bacterium that causes severe infections in the urinary tract, skin and respiratory tract. It is resistant to a number of key antibiotic drug classes and particularly affects people who are immunocompromised. It is often spread by medical equipment such as catheters that are not properly cleaned and is a challenging problem in the hospital environment.
These bacteria have complex protein secretion systems that deliver toxins to inhibit the growth and kill neighbouring cells, thus enhancing the survival of the donor cell. Recent work by an international research group taking place at Diamond has provided new insights into the mechanisms that operate in these systems. The crystal structure of the type VI secretion system in P. aeruginosa was determined using macromolecular crystallography on beamlines I02 and I04. This research will provide new opportunities to examine ways of controlling the effects of these damaging bacteria.
Non-typhoidal Salmonellae bacteria cause intestinal infection and other illnesses in immunocompromised individuals and in young children and are responsible for up to 700,000 deaths each year. Although cephalosporins are the first-line antibiotic treatments there are increasing reports of resistance. A recent study examined the development of antibiotic-resistant non-growing bacteria or ‘persisters’ following antibiotic use. The study characterised the structure and function of toxins formed during the growth of these persisters after they were engulfed by human macrophages. Macromolecular crystallography took place on beamline I04-1 at Diamond. The study extends understanding of the toxin-based growth arrest leading to persister formation in several strains of Salmonella. This will provide opportunities to develop new therapeutic strategies to tackle this damaging bacterial disease.
The ability for pathogenic bacteria to rapidly adapt to their host cell environment is essential if they are to maintain their infectivity. A messenger molecule called c-di-AMP has recently been identified as a vital molecule in this process in Gram-positive bacteria. It controls osmotic stress resistance and virulence, and has also been linked to antibiotic resistance and sensitivity. c-di-AMP is produced and degraded by dedicated enzymes and the activities of these enzymes are tightly regulated in the cell for optimal bacterial growth.
In the human pathogen Staphylococcus aureus, c-di-AMP is produced by the di-adenylate cyclase enzyme DacA. A recent study investigates the activity and regulation of this enzyme in the effort to devise strategies to inhibit di-adenylate cyclase enzymes, as inhibition of c-di-AMP production will negatively impact bacterial growth and also re-sensitize methicillin-resistant S. aureus (MRSA) strains to β-lactam antibiotics. The study determined the atomic structures of the S. aureus enzymes DacA and GlmM and show that GlmM readily binds to DacA in vitro and blocks its activity by masking its active site. Research took place on Diamond beamlines B21 (Small Angle X-ray Scattering) and I03 (Macromolecular Crystallography). This work provides important insight into possible ways to stop c-di-AMP production in bacterial cells which may lead to the design of new antibacterial therapies.
Over recent years, researchers from the University of Southampton and Diamond have been working on an entirely new approach to tackling bacteria. The team brings together expertise in microbiology, biochemistry, and structural biology to aid a fuller understanding of biofilms which are large collections of bacteria that cluster together to create a thick and slimy mass. Bacteria in a biofilm are 100-1000 times more tolerant to antibiotics and are immobile meaning that they are hard to remove. Biofilms are particularly responsible for chronic infections such as non-healing wounds, and for persistent infections such as those that affect people with cystic fibrosis. They also represent health threats through growth on implants or catheters.
The research team have been looking at the mechanisms that regulate the formation and dispersal of biofilms so that antibiotics are able to destroy the individual bacteria and stop chronic infection. Recent data on the cell surface receptors of Pseudomonas aeruginosa proteins were collected on beamline I02, I04 and I04-1 at Diamond. This important work on a new approach to tackling bacterial infection is ongoing.
Another approach in development is the rational design of antimicrobial nanocarriers – tiny lipid-based molecules that protect and deliver bioactive molecules. These would have reduced toxicity to cells and would provide new alternatives as antibacterial resistance develops around the world. At present, most of the commonly used polymer-stabilised nanocarriers are cytotoxic. However, a new study has designed a novel nanocarrier for the antimicrobial peptide LL-37 which promotes cell proliferation and improves wound healing.
The study included use of the I22 beamline at Diamond using small angle X-ray scattering and diffraction. The study highlights the potential for the nano-carrier to deliver antibacterial agents to be used in advanced wound healing, and may open up further opportunities in other bacterial diseases.
Penicillin and the wider family of β-lactams have remained the single most important class of antibiotics since their introduction in the early 1940s. The target of these drugs is a family of enzymes called penicillin-binding proteins (PBPs) that are involved in the final stages of bacterial cell wall production. Despite the development of new classes of β-lactams, resistance remains a global problem.
Mutations in PBPs are a key factor in the development of resistance and are becoming more common. New research from the universities of Warwick, Oxford and Cape Town has identified a new crystal structure of PBPs from Haemophilus influenza, alongside improved resolution of PBP structure from a number of other bacterial pathogens including E. coli, P. aeruginosa and N. gonorrhoeae. These new structures allow researchers to map locations of known resistance mutations for each species. Crystal structures were obtained from studies on beamlines I03 and I04 and it is hoped that this structural approach will lead to further insights into the mechanism of action of these widely reported but poorly understood mutations.
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