Neutrons – A New Weapon Against Cancer
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Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. The development of cancer is a complex process, which can result from minor mistakes in an otherwise well-functioning network of cellular processes. As such, it is essential that scientists have a clear picture of biochemical processes at an atomic level in order to gain an in-depth understanding of how various cancers occur and develop. One technique starting to gain recognition as a powerful tool is neutron crystallography. This is because it allows researchers to visualize the atomic structures of proteins, including the positions of the hydrogen atoms – key players in nearly all biochemical reactions and processes. In addition, neutrons do not damage the samples, so they allow structures to be determined at room temperature, close to physiological temperatures. This is in contrast to more common analytical tools, such as X-rays, where location of the all-important hydrogen atoms is generally not possible, and data collection is complicated by issues of radiation damage to the samples that can lead to alterations to the structure.
A recent study of galectin-3, a member of the galectin family of proteins, exemplifies the ability of neutron crystallography to enhance our understanding of proteins implicated in various cancers. Galectins are carbohydrate-binding proteins that attach to other proteins via the carbohydrates on their surface. When galectin-3 is overexpressed it can enhance the adherence of breast cancer cells to other cells in the human body, suggesting it has a role in tumor development, and the way that cancer spreads through the body in a process called metastasis.
Hydrogen bonds to lactose in the C-terminal carbohydrate recognition domain of galectin-3 as determined with neutron crystallography. The bonds are represented here by light-blue dotted lines. Institut Laue-Langevin
In collaboration with Lund University (Sweden), research carried out using the LADI-III instrument at the world’s flagship neutron facility, the Institut Laue-Langevin (ILL), as well as neutron instruments at Oak Ridge National Laboratory (ORNL) and Heinz Maier-Leibnitz Zentrum (MLZ), focused on understanding how galectin-3 binds to specific carbohydrates. Using neutron crystallography, we were able to visualize, for the first time, the key binding interactions in the protein’s active site, namely the hydrogen-bonds. The results of the study, published in the Journal of Medicinal Chemistry, are significant because if galectin binding to malignant cells is a primary culprit in the progression of breast cancer, a drug that acts as an inhibitor of this binding may be effective as a therapeutic agent. Thus, knowledge of the key hydrogen-bonding interactions provides us with a better understanding of how to optimize the design of galectin-3 inhibitors.
Until now, our understanding of the binding processes have been based upon structures determined using X-ray crystallography, but even with the most high-resolution structures (better than 0.1 nm) it has been impossible to determine the positions of the hydrogen atoms that reveal the hydrogen-bonding interactions dictating how the sugar-binding occurs. Neutron crystallography, on the other hand, allowed the hydrogen atoms to be visualized even at modest resolutions (around 0.2 nm) at room temperature, and so really is the most powerful technique for revealing such critical interactions.
Another example of recent research taking advantage of the power of neutrons was a study of protein kinase G II (PKG II), from the protein kinase family. This protein, if not activated, can increase the risk of stomach cancer, and so researchers are keen to understand the intricacies of how the protein interacts with its known activators, enabling drug developers to exploit this information in future cancer therapy treatments. As published by a global collaboration in Biochemistry, crystals grown by researchers (Andrey Kovalevsky & Oksana Gerlits) at ORNL, using PKG II protein (provided by Choel Kim) from the Baylor College of Medicine in Texas, were exposed to neutrons at the ILL’s LADI-III beamline. The resulting structure made it possible to observe the hydrogen-bonding interactions during the activation of PKG II, providing insights to the enzyme’s activation mechanisms. The results suggest that the activator’s potency depends on its ability to efficiently decrease overall protein dynamics. While further studies are needed to identify how potent activators need to be to bind the strongest, if confirmed, the results will be highly significant for future drug development across a range of diseases – particularly considering ~2% of all human genes encode for protein kinases, and over half of these are linked to various diseases such as cancer and diabetes.
As well as characterizing ordinary cell processes in order to identify drug targets, neutron beams are increasing our understanding of a growing group of cancer pharmaceuticals: the monoclonal antibodies (mAbs). These are being utilized against cancer, as well as other diseases, due to their highly-specific targeting abilities. They can be refined so that they attach themselves to specific protein targets on cancerous or disease-causing cells. Despite the huge potential of mAbs as a therapeutic, they can be difficult to manipulate into a format that can be administered to patients. High concentrations of antibodies are required in each dose to be effective, which results in a solution that is viscous and thus physically impossible to inject. Their tendency to clump together – which is part of their effectiveness in tackling malignant cells – also means that the exact concentration is difficult to predict in solution.
An international collaboration between the ILL, National Center of Neutron Research, University of Delaware and biopharmaceutical company Genentech, used Small Angle Neutron Scattering (SANS) and Neutron spin-echo (NSE) to study the structure and dynamics of mAb clustering. Neutrons are exceptionally capable of analyzing the extremely high-concentrations in the mAb samples, which are required for the high-doses present in the injectable forms. The ILL instruments provide unrivaled high-resolution and neutron intensity, which allowed the researchers to confirm that the inconvenient properties of the mAb solutions came from previously unnoticed small clusters in the solution. NSE is the most sensitive neutron spectroscopy technique. The IN15 Spin-Echo spectrometer has world-leading sensitivity to unravel molecular level motions and interactions. As a result, the potential of mAbs to be used in the treatment of various cancers becomes more achievable, as the administration of the drug becomes more accessible to patients and practitioners.
Image credit: Institut Laue-Langevin. Matthew Blakeley is a research scientist at the Institut Laue-Langevin (ILL) in Grenoble. Graduating from Manchester University in 1999, Matthew completed his PhD in neutron crystallography in 2003, before undertaking postdoctoral research at the EMBL. Since 2007, Matthew has been responsible for the LADI-III beamline at the ILL.