A cell’s surface is made of a lipid membrane that contains many receptors. They can send signals from outside to the cell interior, sometimes to adverse effect, such as inflammation. Numerous drugs target these receptors to reduce such effects without disturbing the cell. In this context, David Alsteens, a researcher at the UCL Institute of Life Sciences, chose an innovative method for identifying characteristics unique to each receptor.
It was in the scientific journal Nature Methods that David Alsteens revealed his new approach to observe—or, even better, decipher—cellular receptors. It relies on a scan by a cutting-edge atomic force microscope (AFM). ‘Microscopy refers to a simple observation,’ Dr Alsteens says, ‘so in a way it’s a poor term, because this device creates a genuine, precise topography of a specific molecule. We can compare how the AFM works to how a turntable’s diamond stylus reads vinyl records. The AFM has a nanoscopic stylus at the end of a pliable lever. The stylus scans literally point by point the surface of the cellular membrane to draw a precise image. Thus it’s possible for us to know the position of a receptor and determine how it interacts with a ligand with which it binds to activate itself. Measuring the strength of these interactions allows us to determine affinity, which is the adhesion force between the ligand and the receptor.’
A unique technique
The AFM has already been used for other purposes. ‘It’s been around since 1986’, Dr Alsteens continues. ‘It was used first on materials, then in ambient air imagery, then in liquid…The technology evolves continually to make surface analyses more precise. More recently, it made possible investigation of micro-organisms and living cells.’ Capable of extremely fine precision, this molecular observation technology is unique.
It also accelerates research. ‘When a pharmaceutical lab has to test a molecule,’ he explains, ‘it has to find the perfect combination of molecule and membrane receptor. To do so, researchers have to test many molecules on a receptor during the first phase of study. They rule out one by one those that don’t work. We, however, look to do the opposite. If it’s possible, using the AFM, to identify more rapidly a compatible candidate, the time the researcher saves is considerable.’
But the AFM doesn’t just provide information. A ligand can be attached to its stylus in order to make contact with the identified receptor. ‘Thus we can measure the forces and interactions between the receptor and its ligand and see how the drug interferes with the connection. We can in a way gauge the competition between the drug and the natural ligand.’ Subsequently, direct contact allows direct access to a specific target and the ability to provoke a reaction. This is particularly valuable when it concerns a drug.
Blood platelet results
Convinced of the AFM’s capacity to contribute to treating pathologies, Dr Alsteens and his team decided to test it on a receptor of blood platelets involved in thrombosis cases: PAR1. He worked with the receptor’s discoverer and winner of the 2012 Nobel Prize for Chemistry, Brian Kobilka of Stanford University (US). ‘Activating PAR1 induces haemostasis, namely the process of blood clotting to avoid haemorrhage. It’s a G-coupled protein receptor, which allows the transfer of information to the cell interior. When one of these cell surface receptors is activated, the G protein attached to the receptor will have either an inhibiting or a stimulating effect on the cell.’
Thus the researchers were able to study the activation mechanisms of this specific receptor. They also observed how it reacts to and interacts with the molecule it activates.
By taking an interest in the circulatory and cardiovascular system, the researchers next tested the effect of a drug intended to reduce heart attack risk: vorapaxar. It blocks the action of the ligand, even if it’s attached to the receptor. ‘So this molecule is very effective, but it poses a problem because it’s insoluble in water. As a result, it has to be encapsulated so that it dissolves in the lipid membrane. Our research aims to go more directly to the target via the aqueous medium, through direct administration at the site.’ This microscopy technique is currently performed on cells placed in optimal conditions in terms of temperature and pH. Dr Alsteens hopes the next step will be to use it in vivo, to see how a virus, for example, interacts with a human cell at the moment of infection.
A multiple-use microscope
The AFM already offers great benefits. ‘For example,’ Dr Alsteens explains, ‘it’s used at UCL by the team of Professor Dufrêne to examine how staphylococcus aureus receptors interact on the skin of different individuals. They’ve found they’re more aggressive on atopic skin.’ Additionally, microorganisms can be attached to the stylus to study their interaction with others and open up new possibilities.
Although found only in research centres, the AFM could one day be used in hospitals, for instance to rapidly identify the structure of cancerous cells. ‘We know, for example, that the most flexible cells are those that most often lead to metastases. This device could rapidly determine what type of cell the oncologist is dealing with and enable him to adjust treatment accordingly.’
A Glance at David Alsteens's bio