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European Biopharmaceutical Review

In the Spotlight

For several decades, X-ray crystallography – in which the scattering of X-rays by the electron clouds of the atoms in a crystalline sample allows the atomic and molecular structure to be determined – has been routinely used by the pharmaceutical industry to determine how their drugs interact with their protein targets. Nevertheless, a limitation of the technique is that hydrogen atoms (with only one electron) are virtually invisible in X-ray analyses, leaving scientists to speculate on their positions despite their importance in drug binding through hydrogen bonding. This is where neutrons come into their own. Rather than being scattered by electron clouds, neutrons are scattered by the atomic nuclei in a crystalline structure, allowing the positions of all atoms to be determined – including lighter elements such as hydrogen. Neutron crystallography is therefore particularly suited for use in drug discovery and development.

Neutron Crystallography

This potential has been acutely demonstrated in a recent study of interactions between a common clinical inhibitor and HIV-1 protease – an enzyme essential for the replication of the virus, allowing it to break polypeptide chains and generate proteins for viral maturation and the production of new infectious virions.

After 20 years of analysis with X-rays, results published earlier this year in the Journal of Medicinal Chemistry, which demonstrated the use of neutrons, provided a more detailed picture than ever before of how an antiviral drug used to inhibit the enzyme actually works. The team were able to identify the position of every hydrogen atom involved in the system, and discover which were involved in the binding of the inhibitor to the enzyme through hydrogen bonding. Critically, the analysis indicated which hydrogen bonds were the weakest and how the binding – and, hence, the drug’s performance – could be improved.

This research represents a major step forward for a technique whose application in the field of pharmaceutical R&D has been held back in the past due to various technical challenges. These included the volume of sample crystals that needed to be grown, the length of time it took for the results to be collected, as well as the lack of dedicated instrumentation available around the world with which scientists could carry out their studies.

During this latest study, the neutron crystallography experiments were carried out on the quasi-Laue neutron diffractometer (LADI-III) instrument at the Institut Laue-Langevin (ILL), where developments in the last decade have addressed the technical challenges. Using the LADI-III, crystals as small as 0.05mm3 can now be analysed – making many more studies feasible.

Developments on instruments such as LADI-III are helping establish a reputation for neutron science as a complement to X-ray studies during various stages of the drug development process. In recent years, neutron scattering techniques at the ILL alone have been the source of a number of major new insights.

Reducing Side-Effects

Earlier this year, scientists from ILL and King’s College London looked at another drug prescribed to HIV sufferers – this time a common antibiotic, Amphotericin B (AmB). This protects against fungal infections – a potentially deadly affliction to those whose immune systems are significantly compromised fighting HIV or going through chemotherapy. While a highly effective drug, the researchers wanted to examine why increased dosages of AmB – prescribed in recent years in response to antibiotic resistance – has started to cause kidney failure and other life-threatening complications.

Using neutron analysis of model membrane systems, the team discovered that at low doses, AmB opens up holes in fungal cell walls – allowing cell material to leak out or harmful material to get in, which kills the cell; however, it is unable to penetrate all the way through the human cell membranes. In the last 20 years, increases in dosage are producing full penetration of both types of membranes, resulting in the damage to healthy tissue observed in trials. By beginning to gain a true picture of how AmB works, scientists can now aim to reduce its side-effects or utilise its therapeutic properties in new, less harmful, alternative drugs.

Nanomedicine

It is not only improvements to existing drugs that are being considered – whole new areas of drug development are being opened up. Earlier this year, researchers from the University of Illinois and the Australian Nuclear Science and Technology Organization used ILL’s neutron-scattering instruments to investigate, on a molecular level, the physical changes undergone by cell membranes as they come into contact with gold nanoparticles.

Gold nanoparticles have been identified by major pharmaceutical companies as a potential future drug delivery agent for the treatment of cancer, based on their ability to target and penetrate cells to transport drugs directly inside the infected tissue. By targeting cancerous cells, this area of research – called ‘nanomedicine’ – aims to reduce or even eliminate the need for surgery.

However, at present we do not understand in any detail the interaction mechanisms between nanoparticles and the cell’s outer barrier – the cell membrane. Without this, it is impossible to determine how dangerous they are and whether their ability to penetrate and destroy cells can ever be harnessed for good, such as in the fi ght against cancer.

The first results from a new project at ILL to better understand these interactions were published earlier this year in Langmuir and looked at how the surface charge of the membranes affects the interaction. Using neutron-scattering techniques, the team showed that depending on a positive or negative surface charge, the interaction with the gold fl akes could produce a protective shield effect, or the complete penetration and destabilisation of the membrane. These are vital insights for the development of future nano-treatments.

Shedding New Light


With the case for the use of neutrons starting to build, there is the potential to revisit our understanding of even common drugs, as was the case in the latest study on the HIV inhibitor amprenavir (APV), which has been used clinically for over a decade. In this latest study, its binding with the HIV-1 protease enzyme was analysed by an international team of scientists from Georgia State University, Purdue University and Oak Ridge National Laboratory in the US, and Harwell Oxford in the UK.

Despite its decade of use, it was only following the fi rst analysis with neutrons that APV ’s true binding behaviour was revealed, painting a rather different picture to that inferred from the X-ray studies, which had overplayed the importance of several hydrogen bonds. In fact, the team found only two strong direct hydrogen bonds between the drug and the HIV enzyme.

The findings present drug designers with a set of new sites to try and improve the drug’s chemistry to signifi cantly strengthen the binding, increasing the effectiveness of this and other inhibitors, and reducing the necessary dosages.

Suggestions of areas for improvement include replacing weaker watermediated hydrogen bonds with stronger direct hydrogen bonds to increase the enthalpy of binding. This could be achieved by changing certain functional groups of the drug in order to expel water molecules present in the active site, which at the same time improves the entropy of binding.

Another approach would be to increase the strength of the two direct hydrogen bonds by the creation of what is known as a ‘low-barrier hydrogen bond’, in which the proton is equidistant between the donor and acceptor atoms. This can be achieved by lowering the ionisation constant of the hydroxyl group of the drug to make it similar to that of the amino acids it binds to, via the introduction of a strong electronegative atom such as fluorine.

Drug Resistance

The findings in this latest paper may also help address one of the biggest issues in combating HIV infection – drug resistance. Evolution of the virus over time produces enzyme variants with weakened binding affi nity to the inhibitor – a process that is actually sped up by the introduction of the drugs themselves. One way around this would be to improve the binding of the inhibitor with the main-chain atoms of HIV-1 protease rather than its side-chain atoms, as the main-chain atoms of the enzyme cannot mutate.

Before this latest study, it was thought that the potential for advances in this area were limited, because the hydrogen bond interactions with the mainchain atoms were already very strong. However, this has been shown not to be the case, creating a new avenue for the development of HIV pharmaceuticals much less affected by virus evolution and resistance.

Future Design

X-ray crystallography has played a crucial role in structure-guided drug design for over two decades. While its value to researchers in this fi eld will continue for many years, combining the use of X-rays with neutrons increases the clarity of how drugs interact with their protein targets, providing the pharma industry with a powerful new tool to improve the performance of their products. It is clear that the future of drug design will feature a combination of the two crystallographic techniques that can provide patients with newer, more effective medicines to not only battle HIV infection, but for a whole host of other diseases as well.

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Dr Matthew Blakeley is the Instrument Scientist responsible for the macromolecular neutron diffractometer LADI-III at the ILL, Grenoble, France. After graduating from the University of Manchester, UK, with a fi rst-class degree in Chemistry, Matthew completed his PhD in 2003. He then undertook postdoctoral research until 2007 at the European Molecular Biology Laboratory outstation in Grenoble, after which he took up his current position. Matthew's research interests are neutron crystallography instrumentation and method development, structural chemistry and structural biology.
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