Hepatitis C drug target comes within sight

A multidisciplinary team of researchers in the Department of Biochemistry has solved the low resolution structure of a promising drug target in the hepatitis C virus. The information gleaned will help scientists to design more effective drugs against the virus which puts more than 170 million people worldwide at risk of developing severe liver disease.

Dr Nicole Zitzmann, Dr Catherine Vénien-Bryan and D.Phil. student Philipp Luik, together with departmental collaborators in the Scripps/Oxford Laboratory and the Structural Bioinformatics and Computational Biochemistry group, have recently published their findings in the online version of the journal P.N.A.S.1

The 3D electron microscopy structure of the p7 channel.

An estimated 170 million people worldwide are infected with hepatitis C which is spread by blood-to-blood contact. In around 80% of cases, the virus persists in the liver and can cause scarring, cirrhosis and ultimately liver cancer. There is no vaccine against hepatitis C, and currently no hepatitis C-specific drugs are available.

Dr Zitzmann's group is trying to develop drugs against a number of viruses including hepatitis C. The group is particularly interested in a hepatitis C viral protein called p7. 'There is a huge therapeutic need out there, and p7 is definitely a target,' says Dr Zitzmann.

p7 is a small protein that sits within the internal membranes of infected cells. Several individual p7 molecules come together to form a channel that allows ions or other small molecules to pass across the membrane. Scientists know very little about what the p7 protein is doing in the viral lifecycle, but believe that it is required to assemble new virus particles inside cells.

'There is a huge therapeutic need out there, and [the hepatitis C viral protein] p7 is definitely a target.'

A few years ago, the group made a chance discovery of chemical compounds that stop the p7 protein from working. 'Ever since, we've been trying to get any information we possibly could about this additional drug target,' explains Dr Zitzmann.

The research that she and her colleagues have published, showing the three-dimensional structure of p7, represents a substantial step forward for them.

The group has spent several years trying to produce p7 protein in cells in a sufficient quantity to be able study its structure - the first step towards developing effective drugs against it. But p7 is very hydrophobic - it sticks to the membrane and does not like being in water - which makes it extremely difficult to extract from cells.

This is where collaborators from the Scripps/Oxford Laboratory under Professor Paul Wentworth stepped in to assist the group. 'We have fantastic chemists here who managed to synthesise this very hydrophobic protein. It's really at the limit of what is possible. It gave us an edge on our competitors,' explains Dr Zitzmann.

An electron microscopy image of stained p7 channels at about 2500x magnification. Individual channels are highlighted with white circles

An electron microscopy image of stained p7 channels at about 2500x magnification. Individual channels are highlighted with white circles

Armed with a large supply of pure protein, the method of choice for structural studies for researchers would be X-ray crystallography. But the protein's hydrophobic nature made this too difficult. After hearing Dr Zitzmann talk about her work at a departmental seminar, a colleague, Dr Catherine Vénien-Bryan, suggested electron microscopy as an alternative approach.

The technique is far from straightforward, as Dr Vénien-Bryan explains: 'Biological specimens are very fragile and it's difficult to attain high resolution. We had to find ways of preparing the specimen in order to be able to see it because it's very small.' The technique was only possible because the group had pure protein.

Philipp Luik, a student in Dr Zitzmann's lab, worked with Dr Vénien-Bryan on the electron microscopy work. They had to find the conditions under which the p7 channel would assemble into the state it adopts in infected cells. Once they started to obtain images under the microscope, they sifted through thousands to find ones that were of sufficiently high quality. Their meticulous approach paid off. 'This is one of the smallest proteins visualised by electron microscopy,' says Philipp proudly.

Models of the p7 protein molecule fitted into the 3D structure of the p7 channel. Six molecules of the p7 protein are shown in alternating colours so that it is easier to distinguish them, and using 2 different ways of representing the folding of the protein - a ribbon-like or surface representation

Models of the p7 protein molecule fitted into the 3D structure of the p7 channel. Six molecules of the p7 protein are shown in alternating colours so that it is easier to distinguish them, and using 2 different ways of representing the folding of the protein - a ribbon-like or surface representation

Reconstructing a three-dimensional (3D) structure of the channel from the images was another challenging task. The researchers used thousands of images of the channel recorded from several positions and then processed the images to find out the orientation of the channel. Combining these images, they were able to construct a map showing the 3D shape of the channel.

Computational biochemists in the department, led by Dr Philip Biggin, took the work a step further by modelling the structure of a single p7 protein using computer methods and fitting this against the 3D structure of the channel. The researchers have now built up a model of how the individual protein molecules lie within the channel.

The research has thrown light on features of the channel that the group did not expect, says Dr Zitzmann. 'There is a kink and a flower-like opening, so we have a big surface area that is available for interaction potentially with other proteins.'

The structure will help to provide researchers with a much better understanding of how the p7 channel functions and will allow them to exploit it as an antiviral drug target.

'Knowing the structure is absolutely crucial to developing drugs against the virus,' says Dr Zitzmann. 'We already have compounds that block this channel in cells. We need to refine these drugs so that they retain all the antiviral efficacy but are less toxic.'

1. Luik, P., Chew, C., Aittoniemi, J., Chang, J., Wentworth, P. Jr., Dwek, R. A., Biggin, P. C., Vénien-Bryan, C. and Zitzmann, N. (2009) The three-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy. Published online in the Proceedings of the National Academy of Sciences (USA): www.pnas.org/cgi/doi/10.1073/pnas.0905966106





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