Neutron scattering experiments have provided new insights into the origin of the side effects of an antifungal drug prescribed all over the world. The analysis conducted by scientists at King's College London and the Institut Laue-Langevin in Grenoble, and published in Scientific Reports, follows 40 years of debate and could help drug developers reduce these harmful complications.
This is a molecular model of the antibiotic - Amphotericin B.
Credit: Dr. David Barlow, King's College London
Wherever you are in the world, indoors or outdoors, the air you breathe contains fungal spores. Though occasionally linked with allergies, asthma or skin irritations, the majority are easily dealt with by the body's immune system.
A far greater risk is posed to individuals whose immune systems are already compromised such as those infected with HIV, severe burn victims or those having just undergone chemotherapy.
However in the past 20 years there has been a dramatic rise in cases of the fungi developing antibiotic resistance. This has necessitated the prescription of increased doses of AmB which unexpectedly produced severe and sometimes lethal side effects. Tests have shown that at these heightened doses nearly 50% of patients suffer from some form of kidney poisoning. In one study 15% of patients on the drug were forced to go on kidney dialysis. In less common cases AmB can even lead to complete failure of the kidneys, liver or even the heart.
The accepted explanation of AmB as an antibiotic has been that it combines with molecules found in the membrane to create barrel shaped holes which allow cell material to leak out or harmful material to get in, killing the cell.
Whilst fungal membranes contain called ergosterol, animal cell membranes, including our own, contain the more familiar cholesterol. It is thought that AmB reacts more readily with ergosterol to form these membrane holes than it does with cholesterol. This would explain why at low doses AmB can be used to treat infection without harming the patient, however there has so far been no direct evidence to support this hypothesis.Structural analysis by neutron diffraction at the ILL
Like all particles, neutrons demonstrate some wave like behaviour, and when they encounter obstacles whose size is comparable with their wavelength they scatter along well-defined angles. It is this property which allows scientists at neutron sources such as the ILL to analyse the scattering patterns and infer the structure of the material the neutrons have passed through.
The team modelled the two different membranes with layers of lipids, fatty molecules found in all cells, which were combined with either cholesterol or ergosterol. The team used deuteration techniques where deuterium, a heavier isotope of hydrogen which is easily picked out by neutrons over its lighter cousin, is introduced to either the membrane model or the drug, tagging that part of the system to follow it during the interaction.
Using these techniques the team unveiled the first experimental evidence for the theoretical 'leaky holes' resulting from the development of 'barrel-like' structures which formed in both membranes upon the introduction of AmB.
However, the barrels were found to penetrate more deeply into the fungal membranes than the human membranes at the same relatively low dose. The implication is that at such low doses during treatment, the AmB can open up holes in fungal cells but is unable to do so as readily in the human cells. Only when the dose is increased do you get holes passing right across both types of membranes and the resulting damage to healthy tissue.
The underlying cause of this difference in membrane insertion may be down to differences in the angle of insertion, identified for the first time in this paper. When the AmB inserts into the fungal (ergosterol-containing) membranes, it appears to be less tilted than when it inserts into the human (cholesterol-containing) membranes.
The next step for Dr Barlow is to improve the resolution of the analysis in order to identify the exact part of the drug which forms these barrel structures. It is this part that drug developers would need to play with to improve AmB's specificity to fungal cells, or use to develop a whole new drug with less harmful side effects.
Dr David Barlow from King's said: 'These are particularly harmful and potentially lethal side effects. The problem is, despite being developed in the 50's, no-one up until now has been able to experimentally determine how AmB works. This is vital if we are to reduce its complications or use its therapeutic properties in new less harmful, drugs.'Dr Bruno Demé, a scientist at the ILL who worked with Dr Barlow on ILL's D16 neutron diffraction instrument said; 'Neutrons are an ideal tool for studying biological materials and their behaviour as proteins, viruses and cell membranes, all of the order of 1 to 10 nanometres, easily fall within the right size scale. Also neutrons are particularly sensitive to lighter atoms such as carbon, hydrogen and oxygen that make up these organic molecules and can distinguish between them very easily. With the world most powerful source of neutrons in the world, the ILL has a long history of significant breakthroughs in understanding and improving drug delivery and treatment mechanisms.'
Scientific Reports, Published 29 October 2012; DOI: 10.1038/srep00778
Paper and images available on request
The Institut Laue-Langevin (ILL) is an international research centre based in Grenoble, France. It has led the world in neutron-scattering science and technology for almost 40 years, since experiments began in 1972. ILL operates one of the most intense neutron sources in the world, feeding beams of neutrons to a suite of 40 high-performance instruments that are constantly upgraded. Each year 1,200 researchers from over 40 countries visit ILL to conduct research into condensed matter physics, (green) chemistry, biology, nuclear physics, and materials science. The UK, along with France and Germany is an associate and major funder of the ILL.
King's College London (www.kcl.ac.uk) is one of the top 30 universities in the world (2012/13 QS international world rankings), and was The Sunday Times 'University of the Year 2010/11', and the fourth oldest in England. A research-led university based in the heart of London, King's has more than 24,000 students (of whom more than 10,000 are graduate students) from nearly 140 countries, and more than 6,100 employees. King's is in the second phase of a £1 billion redevelopment programme which is transforming its estate.
King's has an outstanding reputation for providing world-class teaching and cutting-edge research. In the 2008 Research Assessment Exercise for British universities, 23 departments were ranked in the top quartile of British universities; over half of our academic staff work in departments that are in the top 10 per cent in the UK in their field and can thus be classed as world leading. The College is in the top seven UK universities for research earnings and has an overall annual income of nearly £525 million (year ending 31 July 2011).
King's has a particularly distinguished reputation in the humanities, law, the sciences (including a wide range of health areas such as psychiatry, medicine, nursing and dentistry) and social sciences including international affairs. It has played a major role in many of the advances that have shaped modern life, such as the discovery of the structure of DNA and research that led to the development of radio, television, mobile phones and radar.
King's College London and Guy's and St Thomas', King's College Hospital and South London and Maudsley NHS Foundation Trusts are part of King's Health Partners. King's Health Partners Academic Health Sciences Centre (AHSC) is a pioneering global collaboration between one of the world's leading research-led universities and three of London's most successful NHS Foundation Trusts, including leading teaching hospitals and comprehensive mental health services. For more information, visit: www.kingshealthpartners.org.
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