Fighting HIV with neutrons

When you think about crystallography, the technique that most often comes to mind is X-ray diffraction. It’s no wonder: in biology, more than 88 000 structures of proteins, nucleic acids, viruses and macromolecular assemblies have been determined using X-rays. But as neutron crystallography has improved, it is becoming increasingly useful in helping to design drugs. Researchers at the Institut Laue-Langevin (ILL) in Grenoble, France, have recently used the facility to understand how an anti-retroviral drug targets HIV.

Since the early days of protein X-ray crystallography around one hundred years ago, the technique has undergone dramatic developments and become widespread. High-intensity X-ray beams provided by synchrotron radiation sources allow data to be routinely collected from tiny crystals just micrometres across and in a matter of seconds (see Cornuejols, 2009).

In contrast, the development of neutron crystallography of large biological molecules has been far less pronounced and its application much less widespread. The main reason for this is that the number of particles per square centimetre per second (known as fluxes) from even the most intense neutron sources are many orders of magnitude less than the corresponding fluxes at X-ray sources.

That meant that until recently, neutron crystallographers needed large crystals and exposure times of months to collect sufficient data. Recent improvements, however, allow enough data to be collected from much smaller crystals in just a few days. That’s still longer than many X-ray-based experiments though, so why would we use neutrons? One of the reasons is the smallest atom in the Universe: hydrogen.

The importance of hydrogen

Most drugs work by binding to a specific enzyme involved in a disease, so that its function is inhibited. A lot of modern drug design therefore focuses on analysing and optimising the interactions between the drug and its target. X-ray crystallography has been the favoured method to unravel these structural details, but X-rays do not generally reveal the position of hydrogen atoms in a molecule. These often play a crucial role in binding through weak yet important interactions known as hydrogen bonds.

In contrast, neutrons can locate the positions of all atoms including hydrogen, and so they provide a powerful analytical tool for analysing drug-binding interactions. Recently, this was demonstrated in a study of the binding between an anti-retroviral HIV drug (amprenavir) and its target enzyme, HIV-1 protease. This enzyme is a key part of the HIV life cycle – it breaks down viral polypeptides to create the proteins needed for the maturation and the production of new infectious virus particles.

Scientists fired neutrons at a tiny crystal of HIV-1 protease bound to amprenavir (only 0.2 mm³ in size) to collect data at a resolution of just 0.2 nanometres. These data allowed researchers to locate the hydrogen atoms in the enzyme–drug complex – and, critically, to identify those atoms participating in hydrogen bonding between the drug and the enzyme.

Better resolution

Using previous X-ray studies, scientists had speculated that several hydrogen-bond interactions were important in the binding of HIV-1 protease and amprenavir. However, the neutron study revealed that, in fact, only two strong, direct hydrogen bonds exist between the drug and the enzyme (figure 1). This finding shows drug designers new ways to strengthen this binding by subtly modifying the drug’s molecular structure. Hopefully these changes will increase the effectiveness of the drug and reduce the necessary dosage.

For example, drug designers could make the two hydrogen bonds even stronger by adding a reactive atom such as fluorine to the drug. Alternatively, they could add more direct hydrogen bonds, for example by incorporating larger groups of atoms into the structure that would expel water molecules currently found in the binding site.

The unique sensitivity of neutron analysis to hydrogen atoms provides the pharmaceutical industry with a new and powerful tool for structure-guided drug design. Although the value of X-ray macromolecular crystallography will certainly continue for many years because of its higher resolution, using both X-rays and neutrons reveals more clearly how drugs interact with their protein targets and will no doubt improve the efficacy of other pharmaceuticals in the future.