February 15, 2006 (Vol. 26, No. 4)
Sidec’s TEM Unveils Macromolecular Mechanisms in Biological Samples
A proprietary technology utilizing transmission electron microscopy (TEM) to obtain detailed 3-D images of the conformation of individual proteins and macromolecular complexes in biological samples is providing what Sidec Technologies (www.sidec.com) claims is unparalleled new insights into the nature of disease pathways at the molecular level.
The technology, known as Protein Tomography, was originally developed by scientists at the Karolinska Institute (www.ki.se) during the 1980s and has been further developed and commercialized by Sidec for widespread biotech and pharma applications, from studying the molecular nature of disease pathways to validating preclinical models and investigating the activity of candidate therapeutics.
Electron tomography is a well-established procedure that uses TEM to take multiple 2-D pictures of a frozen sample and build these up into a 3-D picture, explains Hans Johansson, CEO. However, using this technique to visualize individual proteins has traditionally been challenging because the dose of radiation needed to achieve adequate resolution at the protein level damages the integrity of the sample. Conversely, using a low-dose electron beam results in a high signal-to-noise ratio, reducing the level of resolution achievable.
Algorithm Filters Background Noise
A proprietary technology utilizing transmission electron microscopy (TEM) to obtain detailed 3-D images of the conformation of individual proteins and macromolecular complexes in biological samples is providing what Sidec Technologies (www.sidec.com) claims is unparalleled new insights into the nature of disease pathways at the molecular level.
The technology, known as Protein Tomography, was originally developed by scientists at the Karolinska Institute (www.ki.se) during the 1980s and has been further developed and commercialized by Sidec for widespread biotech and pharma applications, from studying the molecular nature of disease pathways to validating preclinical models and investigating the activity of candidate therapeutics.
Electron tomography is a well-established procedure that uses TEM to take multiple 2-D pictures of a frozen sample and build these up into a 3-D picture, explains Hans Johansson, CEO. However, using this technique to visualize individual proteins has traditionally been challenging because the dose of radiation needed to achieve adequate resolution at the protein level damages the integrity of the sample. Conversely, using a low-dose electron beam results in a high signal-to-noise ratio, reducing the level of resolution achievable.
Visualizing Molecular Events
The applications of Protein Tomography in drug discovery and development are manifold, Johansson maintains. The technique can be used to compare mutation phenotypes in healthy and diseased patients, animal models, and cells, study receptor complex, such as ion channel formation, and visualize the effect of a therapeutic antibody on target conformation.
Indeed, there are many applications where we can generate hitherto unobtainable data, he suggests. In the evaluation of drug target mechanisms, for example, Protein Tomography is the only tool that can actually visualize the molecular events occurring in tissues; for example, the conformation of unbound receptors, receptors with ligands attached, and the recruitment of signaling molecules.
The technology can also help reduce the attrition rate in clinical trials by helping to validate preclinical cell-based assays and animal model systems. Using Protein Tomography to compare the molecular events in assay cells or animal model tissues with those that occur in human tissues, provides researchers with the ability to determine whether their preclinical systems are truly applicable. This is a major application for Protein Tomography, Sidec believes.
In drug development, the disease mechanism identified in humans must be extrapolated into animal models and cell-based systems for preclinical research, but for clinical trials you move back into humans again, Johansson continues. The failure of drug candidates in clinical trials can occur at least in part because the preclinical assay systems used did not adequately mimic human molecular biology. Data obtained through the use of Protein Tomography can also allow companies and research groups to strengthen their IP position, by bringing unique insights into molecular events into their patentable knowledge-base.
Refining Tomograms
Preparing samples for Protein Tomography involves the same steps used for electron microscopy, with samples of proteins in solution or cell culture/tissue biopsy samples flash-frozen prior to slicing. Immunolabeling and colloidal gold are used to identify target molecules and align the micrographs. A tilt series of electron micrographs is then taken using low-dose radiation, the resulting images are aligned, and a preliminary 3-D reconstruction performed.
Sidecs algorithm is then used to refine and reconstruct high-resolution 3-D tomograms of biomolecules of interest. Proteins or protein complexes are confirmed by features, including molecular size, characteristic features, specific markers or comparative structural data, and the user can then select individual molecules, rotate them through 360, and analyze their conformation in 3-D.
Visualizing Ion Channel Formation
In practical terms the results are stunning, Johansson claims. Protein Tomography has been applied in clinical research to investigate the molecular mechanism behind glomerular capillary permeability in the kidneys and in a test case with AstraZeneca (www.astrazeneca.com), to reconstruct 3-D images of ion channels in situ, both from cell lines and whole-tissue biopsy samples.
The technology can effectively visualize the subunit assembly and structural dynamics of ion channels, resolving subunit composition. In the AstraZeneca project, the resulting tomograms resolved the volume and structural features of the target complex, showing protein conformational states of fully associated channels, and proteins in the process of forming an ion channel.
Studying Flexible Proteins
Protein Tomography is also ideally suited to studying flexible proteins, such as antibodies, kinases, and complement factors, Sidec points out. By their nature, such proteins are not easily amenable to crystallization for study by x-ray diffraction, while the averaging nature of other analysis techniques generates only a mean value of possible protein conformations.
In contrast, Protein Tomography has already been used to study the flexibility of individual IgG molecules with tomograms, demonstrating the different conformations the antibody molecule can take depending on the spatial relation between the two Fab domains and the Fab-Fc domains.
Sidec was founded in 2000 as a Karolinska Institute spin-out, but it has only been since 2003 that the company has focused its business on leveraging Protein Tomography for biotech and pharma applications. Over the last year, particularly, we have really listened to what the industry wants from the technology, so we can maximize its commercial and research potential, Johansson points out. As a result, requests for collaborative projects and contract research have increased by almost 700%.
Since 2003, in-house research at Sidec has focused on industrializing the software tools used for data processing and analysis in terms of speed, automation, and user friendliness. The company also has an extensive research program for improving the resolution in tomograms.
In the near future Sidec hopes to have created a wide acceptance of the technology as a key tool within translational medicine, Johansson concludes. And looking further ahead, we anticipate that Protein Tomography will be used to improve the success rate for a majority of drug discovery projects through better understanding of disease and drug mechanisms at the molecular level.
