Scientists learn to activate hidden genes in microbes, with potential for drug discovery

Drug discovery sounds like something that happens at a lab bench. Although it does involve a fair share of pipetting, the journey often starts not in a petri dish but in backyards, volcanic craters and hydrothermal vents.

Rob Capon is a professor at the University of Queensland in Brisbane, Australia and affiliated with the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark. He is a natural products chemist seeking the next blockbuster drug – but he is really more of a microbe treasure hunter.

“Microbes cannot bite each other, and they do not carry shells they can hide in – so they tend to slug it out with chemistry,” Capon explains. “You have a billion years’ worth of chemical warfare,” and in some cases these chemical weapons can solve human problems, from disease to pest control.

Now, a new discovery has Capon rifling through all his old finds to check for overlooked treasure – researchers have developed a technique to activate silent clusters of genes in microfungi and some other microbes. Their results, published in the Journal of Natural Products, demonstrate that the technique can reveal new-to-nature chemical structures that could have medical applications.

Low-hanging fruit and secret weapons

The gold rush of natural products science ran from about the 1950s to the 1980s, Capon says – “I liken it to going into a new gold field and picking the nuggets out of the stream. You did not need a shovel, you did not have to turn over any dirt. It was all the low-hanging fruit.”

That boom provided countless medications and products we rely on today: spinosad, an organic pesticide for crops, was isolated from sugar cane in 1985; ivermectin, the antiparasitic agent, was plucked from soil outside a golf course in Japan in the early 1970s; antifungal agents cropped up left, right and centre; and even statins, one of the most frequently prescribed drugs in the world, got their start in a blue-green mould in a Kyoto rice shop.

Those halcyon days are over, Capon says, and now natural products chemists have to do some serious sifting to find as-yet-undiscovered microbes with potential applications. That could mean scooping up extremophile bacteria that live on hydrothermal vents at the bottom of the ocean or taking scat samples from cryptic rainforest creatures.

But finding the unusual microbes is not enough – you have to convince them to cough up their secret weapons.

By the early 2000s, when sequencing a microbe’s entire genome became inexpensive enough, “it became obvious that there are a whole bunch of extra gene clusters in there for which we were not seeing the molecules produced in standard laboratory conditions,” Capon explains. “This microbe’s only supposed to make five different chemistries, and it has 25 different types of biospecific gene clusters – what’s going on?”

Over billions of years, microbes have developed an arsenal of chemical weapons stored in their genome – but they only deploy these weapons when they are needed. Geneticists have tried to copy and paste these silent gene clusters into other systems to activate them, but Capon says that this strategy generally yields these target molecules in only miniscule quantities.

Instead, as a traditional chemist, Capon prefers to “get the microbes that originally acquired the skill and convince them to turn it back on for me.”

A key in a lock

Capon’s current research trajectory started with a trip to the beach.

A graduate student in Capon’s lab convinced his mentor to let him tag along with another group’s field expedition to Heron Island in the Southern Great Barrier Reef, vowing to collect soil samples from remote beaches.

The graduate student came home with a tan and some less-than-impressive scientific spoils. “It was like the magic beans from Jack and the Beanstalk,” Capon recalls. “I gave you USD 2,000 and you give me three little baggies of sand? For goodness’ sake!”

“The truth is that those three baggies of sand turned out to be absolutely brilliant,” Capon says. The researchers found within them two microbes locked in an ancient battle that would inspire more than a decade of research.

By themselves, the two microbes, a bacterium and a fungus, had boring chemistry. But put them together and the petri dish erupted in bright red.

Careful study revealed that “the fungus was producing a very weak antibacterial – a useless little molecule that could barely kill bacteria but was enough to irritate them.” In response, the peeved bacterium began amping up its own chemical defences by producing nitric oxide, a compound that would affect the transcription of its antifungal genes.

But to the researchers’ surprise, the nitric oxide seemed to act like a key in a lock for the fungus as well. “The fungus shut down the biosynthesis of everything except its antibacterial agent,” Capon says.

“We learned through that whole study that nitric oxide was an unexpected player in controlling some of these silent biosynthetic gene clusters,” Capon says. Capon and his team could not help but wonder – could nitric oxide work for other microbes?

Panning for bioactive gold

Over the next several years, Capon and colleagues developed a technique called nitric oxide–mediated transcriptional activation (NOMETA) to force microbes to bring out the big guns.

“You may have seen old black-and-white movies where someone has a heart condition,” Capon says. They clutch their chest, “grapple for a pill, stick it under the tongue and suddenly feel better.” This “ancient angina medication”, nitroglycerin, works by releasing nitric oxide. “This stuff is cheap as anything, so we just add it to the cultures.”

By 2019, the researchers had confirmed that nitric oxide “turns on the production of chemistry in a good number of fungi and bacteria.”

But to pan for drug discovery gold, they needed volume – they would have to pit thousands of old samples against nitric oxide and then meticulously catalogue each compound the microbes produced to check for novelty.

Capon says that this herculean task is only imaginable due to the Global Natural Products Social Molecular Networking platform, a system that enables researchers to upload raw mass spectrometry data and compare the readings with a vast database of known compounds.

“They use very clever algorithms and data analysis and visualisation such that today, with a three-minute run on a routinely available instrument, we can map the entire metabolome, or all the small-molecule chemicals, of a microculture,” Capon explains. “We can do this on a fraction of a microgram of material – very quickly, very cheaply, very robustly.”

“So we then needed to figure out how to do the cultures in small scale,” Capon says. Capon and his team developed a special set-up that would enable them to test microbes from the field at extraordinary volume. “We could do 1000 cultures in a space that would normally be good enough for maybe a dozen or so.”

Uninvited dinner guests and carbon skeletons

With the new culture system in hand, Capon and his team decided to put NOMETA through its paces. But this time, they were not testing some exotic sample – for Capon, these microbes could not be closer to home.

“A termite nest ate big chunks of my house” in Pullenvale, Queensland, Capon explains. “It caused me a lot of grief.” When he first discovered the uninvited dinner guests, “the nest stood about half a metre tall – a muddy sort of mound, like concrete. I drilled a hole in the top and poured in Terminal, which is a termite killer.”

After about a year of home repairs, Capon remembered the termite ghost town and decided to “bust it open,” he says. He was rewarded with a colourful mixture of fuzz, threads and bulbous lumps – a rich ecosystem of unfamiliar fungi. “I stuck it in a big bucket and brought it to the lab.”

Using NOMETA and Global Natural Products Social Molecular Networking, the researchers homed in on a microbe that seemed to produce unusual chemistry in the presence of nitric oxide – a suite of four compounds Capon’s team dubbed pullenvalenes in honour of the partly devoured suburb where the fungi were discovered.

The pullenvalenes are characterised by a triterpene carbon skeleton, a new-to-nature arrangement with as-yet undiscovered properties when acting in a biological system.

“When we isolate a molecule from a microbe, that is the end result of an evolutionary process that has designed proteins, assembled them in production lines and created a genetic element that has been preserved for eons,” Capon says. “Clearly, the capacity to make that chemistry has been so beneficial that that lineage of microbe has hung onto it.”

Applications and chemical spaces

When encountering a new-to-nature chemistry, Capon and his team run through a standard battery of tests to check for superpowers – the new compound is tested against human cancer cells and antibiotic-resistant bacterial strains.

The pullenvalenes did not show interesting activity against these targets, the researchers found. But a negative result on these first batch of tests is not the end of the road for a unique compound.

“Even though we might not know what the biological purpose of some of these molecules is, that reflects our lack of understanding and not the lack of value,” Capon says.

“These days, we are looking at more elegant assays in which we are not interested in selective cytotoxicity,” or killing some cells while sparing others, Capon explains. “We are trying to control Alzheimer’s disease, we are trying to control inflammation. We are looking for better pain drugs.”

Currently, Capon and his team have several irons in the fire – one project is looking for natural products to help to control livestock parasites and stamp out heartworm in cats and dogs. “We also just spun out a company to develop a new anti-inflammatory agent for irritable bowel disease from a human gut microbe isolated from healthy people’s poo,” he says.

“The more you understand about the chemistry that nature has considered valuable, the more you have a clue as to the chemical spaces in which you might seek these molecules,” Capon explains. Otherwise “we are just whacking atoms together and hoping like hell they are going to be bioactive.”