In Australia’s arid bushlands grows an unassuming shrub with an unassuming name—the tar bush, noteworthy to the passing traveller only for its delicate, trumpet-shaped flowers that range in colour from a fiery red to pastel yellows. But the tar bush’s waxy leaves hold wonders.
For millennia, Australia’s indigenous First Peoples have used the tar bush’s close cousins in the genus Eremophila for medicinal and spiritual purposes. Now, new research from collaboration between the University of Copenhagen and the University of South Australia has led to the isolation of compounds previously unknown to science from the tar bush’s leaves that could inspire the next generation of drugs to combat high blood glucose.
Dan Stærk, Professor in Natural Products Research at the Department of Drug Design and Pharmacology of the University of Copenhagen, does not believe in reinventing the wheel. Rather than dreaming up “imaginary” compounds for potential new drugs and struggling to synthesise them in the lab, Dan Stærk and his Natural Products Research Group are bioprospectors, riffling through the catalogue of chemical compounds developed by nature to find candidates for drug development.
“What we do here is use Mother Nature’s very long evolution, in which nature has synthesised a multitude of complex compounds, not in the chemical laboratory, but by a biosynthetic laboratory encoded in the plants’ genes,” says Dan Stærk.
Dan Stærk knew from analysing samples in the University of Copenhagen’s collection that Eremophila plants are rich in diterpenoids, a class of compounds that previous studies suggest could help reduce high blood glucose. But would the tar bush live up to its cousins?
Targets for the tar bush
The Group’s collaborators in Australia removed the tacky resin from the tar bush’s leaves in an acetonitrile solution, a high-tech take on the tea-like decoctions that Aboriginal peoples prepare from the closely related desert fuchsia plant.
Back in Copenhagen, tests of the tar bush extract confirmed that something in the plant could affect the metabolism of carbohydrates – and thereby blood glucose levels – through two cellular targets.
The first is alpha-glucosidase, an enzyme in the small intestine that breaks down the complex carbohydrate we eat – the foods we call carbs, such as pasta and rice, as well as sugar, starch and fibre – into small glucose molecules that can be absorbed into the bloodstream and ferried all over the body for cells to use as energy. Limiting the activity of alpha-glucosidase can reduce the amount of glucose entering the bloodstream after a meal.
Dan Stærk notes that some already approved drugs target alpha-glucosidase, but their gassy side-effects make them untenable as a long-term treatment option for people with high blood glucose. If carbohydrate is not broken down in the small intestine, Dan Stærk explains: “All this sugar-rich material is now in our lower intestines, where the bacteria are having a party with them.”
”The result is extreme flatulence, diarrhoea and stomach pain” to an extent Dan Stærk describes as unliveable. “We are aiming at a drug with another inhibition profile, so that these complex carbohydrates are degraded differently within the intestines,” he says.
Doors stay open
The second target the tar bush resin inhibits is PTP1B, an enzyme that functionally closes the cellular doors that insulin opens.
After a meal, the pancreas releases insulin into the bloodstream. Insulin attaches itself to a special binding site on cells (aptly named the insulin-receptor kinase), causing the end of the receptor protein inside the cell to change shape. Think of it as flipping up the red arm on the mailbox – you’ve got glucose! This triggers a cascade of signals within the cell that ultimately causes transport proteins to move to the cell membrane and invite glucose inside.
But also milling around in the cell is PTP1B, which resets the insulin-receptor kinase to its original state – flipping the metaphorical mailbox arm back down and signalling the transport proteins to close. If there is enough insulin to outcompete the PTP1B, the doors stay mainly open, allowing more glucose to exit the bloodstream and enter the cells.
By suppressing the activity of PTP1B, researchers hope to turn the tide in favour of the insulin. A 2002 study found that mice that had the genes for PTP1B knocked out – eliminated from their DNA before they were born – were resistant to obesity and more sensitive to small amounts of insulin.
Precision over brute force
But what within the tar bush resin inhibited alpha-glucosidase and PTP1B? The researchers separated the candidate compounds using high-performance liquid chromatography, a technique that filters a solution based on polarity, in which molecules with high polarity (easily dissolved in water) travel along in a stationary column at higher speeds than apolar molecules (easily dissolved in oily solvents). The resulting peaks comprise only one compound each.
Testing each compound’s interactions with the target proteins revealed the culprits – two diterpenoids that suppressed PTP1B and one that inhibited both PTP1B and alpha-glucosidase. Although one of the PTP1B-suppressing compounds had been previously described, the other two were unknown to science.
Dan Stærk explains that, although the inhibitory effect of the new diterpenoids is modest, he is seeking surgical precision and not brute force.
“The problem is that PTP1B is a phosphatase” – an enzyme that snips phosphate groups off other proteins to trigger a change in the cell – “and we have many other phosphatases in our body that we do not want to interfere with,” he says. “More important than having a very strong inhibitor, it should have high selectivity towards PTP1B compared with other phosphatases.”
Dan Stærk says that the most exciting finding from the study is that two of the inhibitory diterpenoids have a distinctive structure – a double hydroxylated aromatic ring and no C-19 carboxyl groups. “Rather than saying that our compounds are promising drug leads, we would say that they add information to the structural features that are important in finding potent drugs with specificity for PTP1B and another side-effect profile for alpha-glucosidase.”
Indigenous knowledge and the Nagoya Protocol
Dan Stærk is eager to distance his bioprospecting work from biopiracy. “Many large pharmaceutical companies have historically harvested plants in low- and middle-income countries in the tropics, developed new drugs and earned money from this without giving anything back to the countries that own the biological resources,” he says.
In 2014, Denmark ratified the Nagoya Protocol on Access and Benefit Sharing, an international agreement on genetic resources designed to prevent just that. Under its terms, foreign researchers and corporations must agree to benefit-sharing schemes before they receive permission to conduct research on the genetic resources of the host country.
Arrangements can include investing in conservation, acknowledging traditional knowledge and sharing a stake in the intellectual property rights or royalties from technologies developed as a result of studying the host country’s unique genetics. “The research teams behind this research are of course following the Nagoya Protocol and have legal agreements with the Western Australian government,” says Dan Stærk.
“Because Australia has been separated for a long time from the rest of the world, it is unique – not only the fauna and the flora but also what we call the chemical space,” says Dan Stærk. “It is a treasure chest of new chemical scaffolds.”