Feature: Keeping in front of the flu

This feature appeared in the July/August 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

The initial pandemic scare appears to have come and gone, but swine flu is very much on the minds of many in Australia and around the world. As this magazine goes to print, Australia is in the Protect pandemic phase, indicating the virus still represents a threat to the population. Furthermore, we’re yet to enter the northern hemisphere’s flu season, so we likely haven’t heard the last of pandemic warnings concerning H1N1.

Influenza is also very much on the mind of Professor Mark von Itzstein, who’s name rose to global prominence with the discovery of the anti-influenza drug, Relenza, in 1989. But far from resting on his laurels, von Itzstein continues to look for better and cheaper ways to keep one step ahead of that devious virus.

Von Itzstein is Director of the Institute for Glycomics at the Gold Coast campus of Queensland’s Griffith University and is recognised internationally for his work over many years in glycoscience and drug discovery, and particularly for his leading role in developing zanamivir. This was the first commercially available anti-influenza drug, marketed by GlaxoSmithKiline under the name Relenza. He received the Australia Prize for this discovery in 1996.

Established in 2000 through a multi-million dollar investment by Griffith University and the Queensland State Government, the Institute for Glycomics aims to improve our understanding of the roles of carbohydrates in health and disease. This knowledge will inform the design and synthesis of potential drug candidates and enabling glycotechnologies.

Von Itzstein‘s own research interests remain in the area of infectious disease and drug discovery, using chemical biology as his major investigative tool of trade and carbohydrate-related pathways as the target. Together with a relatively modest-sized group of about 15 people, his work covers a huge range of techniques and approaches from basic biochemistry and particularly enzymology to computational science for drug design and modelling, bioinformatics, synthetic chemistry, virology and, of course, NMR spectroscopy (in their spare time). “We are a very active and focussed group,” he says.

Pathogens go for the carbs

In a biological sense, his group wants to characterise those carbohydrate-recognition phenomena at play on host cells for viruses such as influenza, parainfluenza, dengue and rotavirus, as well as for selected other pathogens like malaria. All of these invading organisms use carbohydrates either to initiate disease or propagate infection and understanding this is a key aspect of the work they do.

“Decoding the language that viruses use for invasion and spread is of course the basis for drug discovery – that is the end game for us. How can intervening in these carbohydrate-recognising processes lead to new opportunities for drug discovery?”

---PB--- Historically, von Itzstein has focussed on sialic acid biochemistry and biology in researching viral disease mechanisms. Sialic acids and sialic acid-metabolising enzymes such as sialidase (also known as neuraminidase) play important roles in the pathogenics invasion of human cells, as clearly demonstrated by the efficacy of antiviral sialidase inhibitors such as Relenza and the later released Tamiflu in reducing the severity and spread of human influenza infection.

According to a 2008 review by von Itzstein, focussed efforts to develop sialidase inhibitor drugs using protein structural information on influenza virus sialidase began in the 1980s, and the development of Relenza a decade later is one of the earliest examples of the successful application of chemical biology and structure-based drug design.

“In the case of influenza virus, we know that both of its surface proteins – haemagglutinin (the ‘glue’) and sialidase (the ‘scissors’) – recognise carbohydrates.”

Based on this fact, von Itzstein’s group is pursuing several research projects centred on influenza biology. The first involves finding out how the haemagglutinin glycoprotein recognises specific carbohydrate molecules on the host cells and how to track changes in these recognition patterns. This work is particularly important in monitoring and unravelling drug resistance and viral mutation and potentially in predicting or circumventing that resistance with novel agents or therapeutic strategies, explains von Itzstein.

“For example, the H5N1 virus, which causes avian influenza, or bird flu, has not yet acquired the machinery needed to recognise the specific human carbohydrates that will allow it to efficiently infect human cells. So, we would like to biochemically interrogate the haemagglutinin of H5N1 and other flu strains at an atomic level to detect those changes when they occur, and ideally to predict when this virus will be competent to infect human cells.”

New approaches to an old problem

Last year, von Itzstein published a technique for making harmless, virus-like particles (VLPs) that have specific influenza haemagglutinin molecules incorporated into them such that, by NMR spectroscopy, his group can start to address these questions in a way not possible previously.

The use of these non-infectious VLP’s allows the carbohydrate-recognition mechanisms to be analysed without the need for high-containment laboratory procedures required for handling ‘real’ influenza virus. According to von Itzstein, the technique also provides a very rapid means to surveil emerging influenza viruses for changes in carbohydrate-recognition patterns and to map the key interactions of carbohydrates with the haemagglutinin protein at an atomic level. “This surveillance approach might also be used to forewarn us of viral mutations that could render the virus competent to invade human cells,” he says.

A second major thrust of the flu work maintains von Itzstein’s long-standing interest in inhibitors of the other viral surface protein, sialidase. His group runs a very strong program in enzymology to investigate the biochemical synthesis and biological evaluation of second-generation sialidase inhibitors with the hope of developing the next phase of influenza medications.

In a worrying trend, reports have emerged of resistance to Roche’s anti-influenza drug Tamiflu, the other commercially available sialidase inhibitor. “Some flu viruses that have appeared around the world recently show significant spontaneous resistance developing, such as one of the standard seasonal human influenza strains of H1N1 (although not the currently infamous strain).”

Fortunately, Relenza is still effective in these cases, but newly apparent resistance to sialidase inhibitors highlights how rapidly influenza viruses can adapt to survive, and underpins the need for a greater arsenal of anti-influenza weaponry to be developed and assembled for broad clinical use worldwide.

To this end, von Itzstein’s group at the Institute for Glycomics have a major programme running that they hope will yield cheaper and effective alternatives to Tamiflu and Relenza, both of which are very expensive drugs to produce. The work involves a lot of kinetic analysis of the compounds to optimise their chemical structures before moving into pre-clinical studies in mouse models, and then, all going well, the agents will be tested in the ferret model commonly used for human influenza drug trials.

---PB--- According to von Itzstein, their drug discovery side of things is progressing very nicely, with some prototype compounds planned to advance into pre-clinical evaluations very shortly, hopefully this year.

“Some of these new compounds target exactly the same spots in the active site of the sialidase enzyme as Relenza, but we have been able to use mimetic agents to mimic the natural substrate recognised by the enzyme, thus the starting materials are much cheaper to produce in the laboratory than the naturally occurring molecules from which Relenza is derived.” They also apply a short chemical synthesis process that will further and significantly reduce end-production costs.

The group’s influenza program is being conducted in collaboration with Institut Pasteur laboratories based in Paris and in Asia. “The Asian connection grew very much out of the prevalence of bird flu there,” he says. “In fact, many of these viruses have their beginnings in Asia, so we thought it was appropriate to work with our Institut Pasteur colleagues there; in Cambodia, Vietnam and Hong Kong. They have very well established facilities to work on the influenza virus story and investigate emerging viruses in the region.”

Keeping one step ahead

“In the longer term, it is clear that we will have to keep coming up with these new drugs continually to fight pathogens such as influenza, which along with bacteria have an enormous capacity for dramatic shape shifting,” says von Itzstein.

To keep one step ahead of them will entail a lot of hard work and energy in terms of designing and developing new approaches as well as looking for new targets on the flu virus itself. According to von Itzstein, there are a few interesting targets coming into the picture now that give hope. It is also possible that combination compounds targeting two or more motifs or regions on the virus will be a better approach that might also reduce the potential for resistance developing.

One thing is clear according to von Itzstein: the interplay between chemistry and biology will continue to play a pivotal role in providing a better understanding of the influenza virus and in designing ways to combat infection.

“We are very excited at what we are finding out about these carbohydrate molecules and their importance in the lifecycle of a virus - irrespective of the type of virus - and the wonderful chance to look at therapeutic and prevention alternatives not considered previously.”

This feature appeared in the July/August 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.