June 1, 2006 (Vol. 26, No. 11)

Tomograms of Proteins In Situ Can Reduce Risk of Late Failures in Discovery and Development

Confidently predicting clinical outcome from research in the discovery phase of drug development remains a major dilemma for pharmaceutical companies. The problem is particularly acute in the transfer of studies of human disease from human biology into model systems and then back again for clinical trials. Unless the true molecular mechanism of disease is understood at the start, the model system chosen for study might not represent or reflect human biology. The risk that such transfer will run into difficulties thus remains unacceptably high.

Sidec Technologies&#8217 (www.sidec.com) Protein Tomography can alleviate this risk. The technique allows 3-D photographs of individual protein molecules or complexes in situ in a cellular context and permits comparisons between species. This proof-of-biology technology thus offers scientists a new means of closing the gap between model systems and human biology.

How Protein Tomography Works

Protein Tomography combines low-dose electron tomography with a unique algorithm that maximizes the signal-to-noise ratio of the tomogram by mathematically filtering the preliminary 3-D construction.

Even in a membrane, Protein Tomography visualizes important classes of proteins in action and at a resolution that locates subunits and shows domain flexibility. Together with labeling by antibodies, for example, the technique now makes it possible to study the subunit composition of ion channels in different cells. Other applications have also confirmed the value of Protein Tomography.

In Situ Ion Channels in 3-D

Ion channels are important membrane proteins and widely regarded as attractive drug targets. As differences in subunit composition and modulator interactions can alter how a channel interacts with molecules developed as drug candidates, understanding the subunit composition in the cell membrane is essential when designing assays and choosing animal-model systems.

In a study with AstraZeneca (www.astrazeneca.com), Protein Tomography resolved the 3-D conformation of an ion-channel complex in its true biological environment and reconstructed its extracellular and intracellular components (Assay and Drug Development Technologies, 2004, vol 2, 5). The channel associated in the form of a 15&#821117 nm high and 9&#821110 nm wide tetramer with a central pore. The study demonstrates that Protein Tomography visualizes in situ protein complexes and thus has a potentially valuable role to play when developing cellular screening assays.

Hormone Receptor Activation

For many years, the growth hormone-induced receptor dimerization model has served as a paradigm for cytokine receptor activation. Many in vitro and in vivo studies supported the formation of a 2:1 dimer:hormone complex. Nevertheless, one developer of screening assays suspected that the dimer could exist alone in the cell membrane. That company, Biovitrum (www.biovitrum.com), turned to Protein Tomography to see if this was true. Figure 1 shows that they are correct; tomograms of the Growth Hormone Receptor (GHR) in a cell (A) and in solution (B) clearly show a dimer in the absence of the hormone.

Within a couple of years, published data supported by FRET, BRET, and more crystallography confirmed this finding and contributed to a revised theory for growth hormone receptor activation. In the meantime, the rest of the Protein Tomography study revealed even more data about the mechanism of action of the growth hormone receptor.

Fig 1: Extracellular domains of the GHR are shown in blue.(A) GHR dimer in a cell membrane (B) GHR dimer in solution (C) x-ray crystallography image of GHR dimer plus GH shown in grey (PBD ID: 3HHR)

Healthy & Diseased Tissue Compared

A recently published academic work, yet one with commercial implications, used Protein Tomography to elucidate the role of nephrin protein in in vivo plasma filtration in the kidney (Journal of Clinical Investigation, 2004, 114, 1475-1483). Nephrin is the key functional component of the slit diaphragm that acts as a filter in renal glomerular capillaries. Healthy and diseased (nephrin-deficient proteinuric patients with congenital nephrotic syndrome of the Finnish type [NPHS1]) tissue from humans was compared at the molecular level. Tissue from rats and mice was also examined.

Tomograms revealed a clear difference in the filter structure between healthy individuals and NPHS1 patients (Figure 2). The healthy slit (A) was wide (approximately 35 nm) and contained the slit diaphragm structure with small pores. Strands were found corresponding to the extracellular part of nephrin (approximately 34 nm long). The slit in NPHS1 patients was collapsed and lacked the nephrin-type strands (width approximately 15 nm), this contributed to a much more disorganized structure, presumably allowing the leakage of plasma proteins.

As Protein Tomography data can be used for comparison between different types of tissue and proteins in solution, further studies were initiated, namely visualizing the structure of full-length nephrin in transfected cells and individual recombinant nephrin molecules in solution.

These tomograms showed that the recombinant nephrin molecules have a 35-nm elongated form. The structural similarity of nephrin in cells and in solution was clear. Similar length strands were found in healthy slit diaphragms, strongly suggesting that they to a large extent consist of extracellular portions of nephrin molecules.

In addition, Protein Tomography data revealed a similar slit diaphragm structure in mouse and rat kidney, showing that kidney filter morphology is conserved in different species. This finding suggests that animal models most probably are relevant systems for further studies of the human biology of the kidney filtration barrier.

By taking 3-D pictures of proteins, Protein Tomography allows researchers to see biomolecules in their cellular context. Tomograms provide insights into the conformation and flexibility of functional targets and their environment.

Protein Tomography data helps elucidate underlying disease mechanisms and can thus help make stop/go decisions earlier in the drug discovery process. It also has the potential to explain the differences between nonresponders and responders at a molecular level. Protein Tomography also supports the development of biologically relevant assays and model systems reducing the risk of failure in drug discovery projects.

Fig 2A: Tomogram of slit diaphragm from a healthy individual; 2B: the absence of slit diaphragm in a patient lacking nephrin expression

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