New alternative oxidase (AOX) compounds developed by Tony Moore, Professor of Biochemistry at the University of Sussex, could have far ranging potential applications to combat a range of fungal infections which are leading to devastating crop losses and life-threatening illnesses worldwide.

The University of Campinas in Brazil (UNICAMP) is collaborating with the University of Sussex to test the use of the patented AOX compounds to combat Witches’ Broom, a fungal disease which has decimated cocoa production in the country, with the long-term goal of developing and commercialising new anti-fungal agents.

Brazil was the second largest producer of cacao in the world until it was hit by a fungal disease called Witches’ Broom, Moniliopthora perniciosa, in 1989, which led to a drop in production of around 70 to 80%. The plant produces a tangled mass of broom-like stems and stops producing cacao fruit after it is attacked by the fungus. “It’s a major problem in Brazil. It has devastated our cocoa farms,” said Dr Mario Barsottini, a joint Research Fellow at UNICAMP and the University of Sussex who is carrying out research in Moore’s lab. “Most cocoa plantations are family businesses. Once the disease reached Bahia (a state in Brazil which produces most of the country’s cocoa), around 200,000 people lost their jobs.”

“It’s incredible to think that something I had envisaged helping boost cereal crop production has the potential to save lives around the world.” – Tony Moore, Professor of Biochemistry, University of Sussex

Barsottini is now carrying out research on how to use the AOX protein as a target for controlling Witches’ Broom and other fungal diseases by testing the Sussex patented alternative oxidase inhibitors against the fungi. If successful, this would further expand the number of compounds already developed by Moore’s lab, which has already produced five AOX agro-chemical compounds which are patented by the University of Sussex (or the patents are pending).

Moore began researching the alternative oxidase, an enzyme located within the mitochondria, over 40 years ago, beginning with plants. He is now a pre-eminent expert in understanding the structure and function of this protein found within the mitochondria of the cell. “It’s an interesting but not well researched protein,” he said speaking from his lab on campus. “Although it had been known for over 100 years, it’s only in the last 20 years that we have had some idea of its structure.”

Many fungicides block a fungi’s ability to respire, but fungal pathogens are adept at developing resistance to treatments. Moore’s early research revealed that the alternative oxidase is an alternative respiratory pathway that exists in plants, some fungi and parasites which becomes expressed when the plant or human fungal pathogen becomes stressed by, for example, drought, infection or exposure to fungicides. This effectively allows the fungi to bypass the fungicides in order to continue to respire and grow. “That was one of the lights that suddenly went on in our brains thinking this is really important. We could produce new compounds that could be combined with commercially available fungicides to inhibit the alternative oxidase.” If they could learn to block the AOX protein, the fungi couldn’t continue to grow.

His lab next identified the structure of the alternative oxidase, which allowed them to develop compounds which prevent the enzyme from functioning. “Understanding the structure of the protein allowed us to design very specific inhibitors,” said Moore. “They are a bit like a lock and key. The lock is the alternative oxidase. So we have designed new types of drugs which are like keys that can specifically inhibit that particular activity of the oxidase.” Crucially, these inhibitors don’t interact with or inhibit any other proteins that are present within the cell.

Global fungicides market is large and growing

The potential market for such compounds extends far beyond Brazil. The global fungicides markets worldwide is large and growing as crops are increasingly resistant to fungicides. Regulators are looking for new, safer fungicides and solutions that will work. According to Zion Market research, this growing market could reach USD $17.58 billion by 2022.

In the last ten years, University of Sussex research has also revealed that AOX compounds could potentially be used in the future, not just on farms, but also in hospitals to combat fungal infections affecting humans. “When we first began thinking about applications for my research, our ideas centred on protecting cereal crops,” said Moore. “But the fundamental problem is the same whether it is as a field of wheat or a human patient – fungi developing a resistance as they are exposed to ever-more potent traditional treatments.”

Parasite causing African sleeping sickness contains AOX protein

The link to human fungal infections was first identified when Moore and his lab collaborated with Professor Kiyoshi Kita at the University of Tokyo and Nagasaki. They discovered that the protozoan parasite which causes African sleeping sickness, Trypanosoma brucei, contains the same AOX protein that Moore’s lab initially discovered in plants. Because this protein is absent from humans, understanding the AOX can help with designing new inhibitors and drugs to kill the parasite by disrupting its ability to survive. The protozoan parasite is totally dependent, in the bloodstream form, on the alternative oxidase pathway to respire, which is why it is of such interest to Moore’s lab. Moore’s team has recently determined the structure of the trypanosomal AOX at the atomic level, and identified the substrate and inhibitor binding site of the protein which would allow them to block it.

Compounds could combat fungus identified as ‘serious global health threat’

The genome of Candida auris, a virulent new form of thrush, has recently been sequenced and Moore’s laboratory found it contains a gene encoding the alternative oxidase. The Mycology Reference Laboratory (MRL) tested the AOX compounds against Candida auris and found that at least one novel AOX compound appeared to be effective at controlling its growth. The fungus, first identified in 2009, is multi-drug resistant and causes multi-organ failure, with patients facing a 33% chance of dying if they contract it. The American Centre for Disease Control and Prevention has called the fungus a “serious global health threat,” while the World Health Organisation has warned that it has the potential to become a worldwide pandemic.

“It’s incredible to think that something I had envisaged helping boost cereal crop production has the potential to save lives around the world.” Moore’s lab is now working to develop inhibitors against Candida auris to be used for clinical applications.

Moore’s lab is also collaborating with Finnish researchers to determine if the AOX protein itself could help protect against septic shock through gene/protein therapy, where the protein is introduced into the cell. It could also potentially treat those with mitochondrial diseases, which result in neurological conditions such as Alzheimer’s and Parkinson’s disease. Given the rising prevalence of fungal infections all over the world and the growth of ageing populations, the clinical market for the compounds is also set to grow. Moore is now setting up a spin out company to further develop the compounds into pharmaceutical treatments and drugs.

The potential range of applications related to the AOX protein is clearly enormous. In fact, there are so many potential uses for the AOX compounds that Moore refers to it as “platform technology”. “The number of organisms that we are now recognising contain this protein is proliferating,” said Moore. “There’s a whole tranche of different organisms that contain it.”

Compound could have important environmental and commercial impact in Brazil

Back in Moore’s lab, Barsottini remains entirely focussed on testing the alternative oxidase inhibitors against the Witches broom fungi. “Using the analogy of the lock and key, with the key being the alternative oxidase inhibitor, it’s hard to predict how the key will fit into the lock. So we have to test different keys and see which is a better fit.”

The potential environmental and commercial benefits of a future AOX compound for Brazil alone would be enormous. Cocoa farms are planted under the shade of native trees. As cocoa production become less financially viable, trees are being cleared to make way for growing other crops. More effective compounds could also reduce the use of fungicides and pesticides. “Brazil is the second largest consumer of pesticides in the world,” said Barsottini. “Lots of the commercial compounds available have been developed for other fungi. We use them, but, as they’re not very effective, we have to use them in large quantities.” The potential economic benefits for the struggling small holder cocoa farmers is hard to quantify, but what is certain is that this miniscule protein could potentially have a huge impact on their lives.

By Suzanne Fisher-Murray, Research Communications Manager, University of Sussex