Bulletin

Issue: July 2004

Research

Working towards a new class of insecticides

A collaboration between researchers from La Trobe University and the University of Queensland could lead to a whole new class of insecticides.

The work focuses on small, cyclic proteins known as cyclotides. Originally extracted from the leaves of a weedy tea plant from central Africa, these unusual compounds have now been found in many widely differing groups of plants, including native Australian violets. But until recently, the function of the cyclotides was something of a mystery.

'We decided to feed cyclotides to caterpillars,' says Professor Marilyn Anderson of the Department of Biochemistry. 'We thought anything produced in such abundance in leaves - where you get 20 to 30 different types produced by one plant - probably had something to do with plant defence.'

Professor Anderson turned out to be dead right. As it happened, she and her team already had a stock of caterpillars they could use. They were of an important pest of cotton and corn, the moth species Helicoverpa puntigera. When fed a diet containing cyclotides, the insects did not grow, and nearly half of them died within a fortnight of hatching.

'In fact, the first feeding trials were aborted because the caterpillars kept escaping when exposed to the cyclotide diets. While those fed on a normal diet would just sit there and eat contentedly, the test animals did all sorts of things to get out.' As a follow-up, the La Trobe researchers are now involved in a series of studies probing just how the cyclotides affect insects, and how and where they are made and stored in the leaves of plants.

Cyclotides first came to notice when Lorents Gran, a Norwegian doctor working with the Red Cross in the Congo in the 1960s, observed a tendency of the contractions of women in labour to accelerate just after they were visited by relatives. He soon tracked down the cause - a traditional medicinal tea brewed from the leaves of a local weedy plant smuggled into the hospital.

On his return to Norway, Gran extracted the active ingredient from the tea. He found it was a small protein, but was unable to unscramble its three-dimensional structure. That did not happen for another 20 years, when Professor David Craik, now of the Institute for Molecular Bioscience at the University of Queensland, began using nuclear magnetic resonance (NMR) at Oxford University to solve the structures of these unique proteins.

When he looked at the first cyclotides, Professor Craik found something so unusual he could hardly believe it. He only felt confident enough to publish the structure a couple of years later. It was a ring of about 30 amino acids - the building blocks of protein - but across the middle were three cross linkages between sulphur atoms, which produced a pretzel-shaped knot. He named these compounds cyclotides after discovering many more examples in plants other than just the African weed.

The compounds have since become a hotbed of research because, not only is the cyclotide structure small for a protein, it is also exceptionally stable. Unlike almost all other proteins, for instance, it resists boiling as well as exposure to acids and organic solvents. In fact, it is so stable, that he reasoned it might be able to pass through the digestive system without being broken down, and yet it is so small, it should be able to be absorbed easily.

Such a molecule, Professor Craik argued, could form a useful platform for oral drug delivery. And having determined how to make cyclotides synthetically, he and his group at the University of Queensland are now working on this very possibility.

The La Trobe group became involved when Professor Craik and Professor Anderson, with whom he was working on another project, began to talk about what the compound actually did in plants in the first place. The very properties that make cyclotides useful for drug delivery, however, also make them very difficult to work with. They are tiny and they are soluble in water, organic solvents and acids so they are often difficult to detect.

Research assistant, Barbara Barbeta is observing the guts of insects under the microscope to see what happens after cyclotides have been consumed. In order to determine the impact of cyclotides, Ms Barbeta has to understand what a normal caterpillar gut looks like. 'There's not a lot of work been done on this,' says Professor Anderson, 'even though we are using some of the worst insect pests of cotton in the world - major pests that we spend billions of dollars on controlling.'

Because such work demands highly specialised methods and equipment the research groups in Brisbane and Melbourne have taken to pooling their knowledge and resources. Postgraduate students working on cyclotides tend to be jointly supervised by Professor Craik and Professor Anderson, and often shuttle between the two laboratories.

For instance, while the chemical work on the structure of the cyclotides is the preserve of the Queensland team, there is an overlap with the studies of Professor Ander-son's group on just how the molecules are produced and function in biolo-gical systems.

It was a former postgraduate member of Professor Anderson's team, Cameron Jennings, who first showed that the cyclotide molecule is formed as a unit, the product of a single gene. This protein is then processed and folded in the cellular membranes, known as the endoplasmic reticulum and the Golgi apparatus. But just how that occurs, and even where the molecule ends up, are still unanswered questions. And the process may be different in different plants, even though the actual end product itself is exactly the same.

In order to study just how the compound is made, Professor Anderson's group is making a series of transgenic plants incorporating different mutants of the cyclotide gene, and observing how they are expressed. 'We want to see what affects the assembly process.'

But the Australians are not the only ones working on cyclotides. Already, says Professor Anderson, a company has been established in Germany to produce cyclotides on a large scale, and companies in the US are also taking interest. 'We once had the whole area to ourselves, now we have some competition to keep us on our toes.•

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