Scientists mine for potential drugs in the Berkeley Pit and other industrial sites

Like most good mysteries, it began on a dark and stormy night.

On the morning of Nov. 14, 1995, residents of Butte, Montana, found hundreds of dead snow geese floating in the runoff and groundwater that had flooded a copper mine at the edge of town. A squall the night before had forced an emergency landing. When they were found, burn sores dotted their bodies, and their white feathers were dyed a sickly, jaundiced orange.

What happened to the birds wasn’t actually a mystery. They’d had the misfortune of landing on the toxic waters filling the remnants of what used to be one of Montana’s richest copper mines, the so-called Berkeley Pit. Now a Superfund site, meaning it’s designated by the US Environmental Protection Agency for toxic waste cleanup, it was filled with a deadly broth that had a pH (2.5) rivaling that of sulfuric acid. Water bubbling up from old mine shafts had seasoned the pit with copper, cadmium, and arsenic.

What was a mystery is how, months later, Andrea and Don Stierle found fungi and other microbes thriving in the pit, an environment so harsh and extreme it killed birds within hours. Nothing, experts said, could live here.

The Stierles had their reasons for looking. First, scientists were discovering life in other seemingly uninhabitable places, like undersea vents and the simmering rainbow cauldrons at Yellowstone National Park. Why not the Berkeley Pit? Second, the pit was literally in the Stierles’ backyard. They were then on the faculty at Montana Tech and didn’t have the grant money to look anywhere more exotic. And third, they knew organisms living in extreme environments like the pit would likely synthesize some rather interesting molecules. The same chemicals that were keeping these organisms alive under such crazy conditions might also keep humans alive when facing harsh maladies like cancer.

Their “What the hell? Let’s give it a whirl” attitude paid off as soon as they brought their samples back to the lab. Within days of streaking the pit water on the nutrient-rich agar that coated their petri dishes, they isolated several fungi. One was a yeast that arrived in the pit on the bodies of the snow geese and somehow survived.

“Most microbiologists told us yeasts do not grow at pH 2.5, so you did not find a yeast,” Andrea says. “I thought, ‘Well then, what is this?’ ”

Many scientists have been investigating the planet’s countless extreme environments, on the hunt for molecules that will inspire the next blockbuster drug or technological breakthrough. With some scientists estimating that less than 5% of fungal species and 1% of prokaryotic species on Earth are known, a lot of biodiversity remains to be explored.

The Stierles, now at the University of Montana, run one of the few labs looking at human-made extreme environments on this same quest. From abandoned copper mines in Montana and Vermont to a coal seam in Kentucky that’s been burning for half a century, natural product chemists have begun to identify potential new pharmaceuticals in the most unlikely of places.

“Extremely hostile environments are an evolutionary playground,” says Tomasz Boruta, a bioprocess engineer at Lodz University of Technology, in Poland. “Organisms need to evolve all sorts of new compounds to adapt to those harsh conditions.”

The researchers face an uphill battle long before they enter the competitive world of big pharma. For one, growing some of the extremophiles—organisms living in these environments—back in the lab can be difficult, if not impossible. Scientists can’t collect molecules from difficult-to-culture microbes readily. Drug discovery, then, must proceed by examining an extremophile’s DNA and searching for the telltale cellular machinery that makes such molecules. What’s more, selecting and identifying any promising molecule remains an expensive and arduous process.

Still, the chances that these tiny survivor organisms will yield useful molecules is high: the compounds they synthesize have to be potent and unique to help keep them alive. Though the work has yet to arrive at a doctor’s prescription pad, experts say that it’s probably only a matter of time until some of humanity’s biggest disasters begin to yield some lifesaving compounds.

“Microbial diversity is largely untapped,” says Barry O’Keefe, chief of the Natural Products Branch at the National Cancer Institute. “These new structures can inspire new syntheses. It pushes biology forward and it pushes chemistry forward.”

Microbial mining

Some of humanity’s oldest and most common pharmaceuticals originated in the natural world. As exemplified by aspirin, morphine, quinine, and penicillin, we have learned to harness the small molecules that plants and microbes use to fight off competitors, predators, or pathogens. These compounds—called secondary metabolites because they’re not directly involved in an organism’s growth, development, or reproduction—can be found in microbes living everywhere from the top of Mount Everest to the bottom of the Mariana Trench. Although useful drugs can be found in less extreme settings—the antibiotic streptomycin came from a soil bacterium—beginning in the 1990s, natural product chemists have increasingly turned to some of the world’s least hospitable habitats in search of the next lifesaving drug.

Unlike the microbes that swarm about balmy coral reefs or the bacteria abundant in our own guts, organisms living in highly toxic environments tend to exist at a low population density. Because microbes typically produce secondary metabolites to outcompete their neighbors, researchers originally thought that fewer neighbors would mean reduced competition and, thus, far fewer secondary metabolites, explains Marcel Jaspars, an organic chemist at the University of Aberdeen, in Scotland. Instead, the opposite is true.

“Resources are few and far between, so microbes have to fight even harder” in extreme environments, he says.

The Stierles learned this firsthand when they shifted their work from biodiverse tropical reefs to the Berkeley Pit. As postdocs at the Scripps Institution of Oceanography, Don and Andrea began their careers by exploring deep-sea life off the coast of Bermuda for potential new compounds. Like many researchers around the world, the Stierles learned that many of the bioactive molecules found in large, multicellular organisms were actually produced by the microbes living in and on the organisms’ bodies.

Take the breast cancer drug Taxol. Scientists first isolated it from the inner bark of the Pacific yew tree, but in 1993, as newly minted professors at Montana State University, the Stierles showed that one of the tree’s symbiotic fungi could also biosynthesize the compound.

Despite publishing a breakthrough Science paper on their Taxol discovery, the Stierles’ funding dried up in the mid-’90s. When they heard some geology colleagues were going to be out working in the Berkeley Pit, less than a mile (1.6 km) from their lab, the Stierles asked them to take samples. Swabbing some pit water onto agar plates was cheap, and it would scratch their scientific itch as to whether anything could live in the toxic stew.

Their near-immediate discovery of fungi in the pit made them forget about Bermuda and focus their efforts entirely on the extremophiles in their own backyard. Using a nutrient broth seasoned with pit water, Andrea could culture enough of the fungi to screen whether they produced potential antibiotics. When her search turned up empty, the Stierles began to think about the types of secondary metabolites that microbes would have to produce to thrive in such a hostile world. These microbes didn’t need antibiotics to attack their rivals. For them, the main focus was just to survive the low-pH environment and the toxic metals surrounding them. To do that, the organisms were surely turning on a protective chemistry cascade of some sort.

“We realized that the compounds they made in that particular environment are the kinds of things that we might be able to use as drugs for our toxic environments, which are cancer and inflammation,” Don says.

To focus their efforts, the Stierles screened one of the fungi they had isolated—a variety of Penicillium rubrum—for compounds that would inhibit matrix metalloproteinase-3 (MMP-3) and caspase-1. MMP-3 breaks down proteins in the structural and biochemical support system of cells known as the extracellular matrix. Scientists think it helps cancer metastasize. Caspase-1 is a protein that helps trigger inflammation.

The researchers used chloroform to extract potential compounds from their fungal culture and tested the crude extract for its ability to inhibit either caspase-1 or MMP-3. After several rounds of purification, analysis of the extract by chemical ionization mass spectrometry revealed two new hybrid polyketide-terpenoids, which the Stierles dubbed berkeleydione and berkeleytrione in a 2004 study in Organic Letters. In tests against cell lines from the National Cancer Institute, berkeleydione showed activity against non-small-cell lung cancer. Further tests have been put on hold for lack of funding.

The Stierles had similar luck 2 years later, in 2006, when they isolated a new spiroketal called berkelic acid that showed activity against ovarian cancer cells. Follow-up studies in 2007 and 2008 identified additional groups of novel compounds: three meroterpenes called berkeleyacetals and four new bioamides, the berkeleyamides. In 2011, when the Stierles combed through old data to see whether they’d missed anything, they found three additional meroterpene berkeleyone analogs, which they reported in the Journal of Natural Products. Today, the scientists are still testing these compounds for their ability to work against specific human diseases.

It’s only been in the last 5–10 years that scientists have been looking at compounds from extremophiles, the NCI’s O’Keefe says. “The field really needs some more time to get their findings off the ground.”

The Stierles’ strategy of identifying bioactive molecules by sequentially purifying a crude extract from a fungal culture worked only because they could readily grow P. rubrum in the lab. Most extremophiles can’t be grown in pure culture. Without this ability, scientists can’t identify them, let alone determine what secondary metabolites they might produce. Advances in genetic-sequencing technology, however, have provided new approaches.

In 1998, a team of microbiologists led by Jo Handelsman, then at the University of Wisconsin–Madison, developed a new technique called metagenomics that allowed the scientists to study microbial DNA directly from environmental samples, without the need to culture an organism in the lab. Previous sequencing methods required a lot of DNA, which meant scientists needed to either grow a lot of the microbe in the lab and extract the DNA or know what species of microbe they were looking for in order to pinpoint and then amplify the organism’s DNA with a tool called polymerase chain reaction (PCR).

Metagenomics eliminated the need for either of these steps, allowing scientists to take small samples of soil or water and directly sequence the DNA without needing to know anything about which species might be there or how to culture the organisms. This revolutionized environmental microbiology and, by extension, natural product drug discovery by allowing scientists to identify the microbes they find and some general classes of molecules the organisms might be making.

“It was so much harder for us to understand biodiversity before metagenomics,” says David Sherman, a medicinal chemist at the University of Michigan.

Middlebury College’s Lesley-Ann Giddings uses metagenomics and other strategies to investigate the extremophiles living in some of Vermont’s abandoned copper mines.

“You can sequence the DNA and have access to all the genes that might produce secondary metabolites, but you still won’t know what will trigger that organism to produce those metabolites,” she says.

By comparing samples taken over multiple years in Vermont, in January and June Giddings hopes to understand how seasonal changes affect resistance to heavy metals and secondary metabolism in the mine microbes. Rapidly identifying the potential functions of secondary-metabolite genes, however, requires scientists to compare the genes they find with similar DNA in databases. Many times, says organic chemist Jon Thorson of the University of Kentucky, researchers will try to identify a microbe by matching its DNA with sequences in a database and come up empty handed, making their work challenging.

“In our soil samples, we see a high percentage of ‘unknown’ microbes in our metagenomic analyses,” Thorson says. But there’s a silver lining, he adds: “This suggests ‘untapped’ microbes with potential for new secondary-metabolite discovery.”

Thorson would know. He has crisscrossed Kentucky, spelunking through the state’s countless coal mines on the hunt for never-before-seen extremophiles and their secondary metabolites. Some of the mines stretch about 11 km into the earth, and when Thorson and his colleagues haul up samples of these denizens of the deep, it may be the first time they have seen the light of day in millions of years. This long period of evolution in relative isolation means that Thorson can find many wild and wonderful new molecules. Since 2012, when he first began searching Kentucky’s old mines for potential new drugs, Thorson has identified nearly 600 pure microbial natural products, 54% of which are novel.

In 2017, Thorson published the discovery of 12 abyssomicin metabolites from Streptomyces species found in the soil collected near a burning coal seam at Lotts Creek in eastern Kentucky (J. Nat. Prod. 2017, DOI: 10.1021/acs.jnatprod.7b00108). One of the metabolites, abyssomicin W, contains a new 8/6/6/6 tetracyclic core; abyssomicin X carries a linear spirotetronate, the first time a naturally occurring version of this structure has been reported. Further analysis of the Streptomyces DNA identified the gene cluster responsible for abyssomicin synthesis.

Thorson’s lab has also identified several new pyranonaphthoquinones from microbes at the Ruth Mullins coal-seam fire site, also in eastern Kentucky. The team showed that frenolicin B, a prototypical member of this family of molecules, has potential anticancer and antimalarial activity (Cell Chem. Biol. 2019, DOI: 10.1016/j.chembiol.2018.11.013).

Sticking with it

For their part, the Stierles have begun to shift their attention partly away from the anti-inflammatory, anticancer compounds that have occupied them for much of the past decade and back toward potential antibiotics. The shift began when Andrea tried to reisolate some of the fungi from her original pit samples, now over 20 years old. (In recent years the pit has become unstable, making access challenging.)

She had trouble getting the fungi to grow separately, in pure cultures. They kept contaminating one another. After months of painstaking work, Andrea succeeded in creating pure cultures of each of the fungi, but she found no new compounds.

Then, with the same mixture of scientific curiosity and adventurous spirit as when she began this work, Andrea decided to grow them together. Historically, scientists have looked for antibiotics by pitting a fungus and a bacterium against each other, the idea being that the fungus might produce an antibiotic to win the battle for resources. Instead the Stierles put two fungi against each other, and the competition yielded secondary metabolites they hadn’t seen before.

“The combined culture did not look anything like either of the individual fungi” in terms of the metabolites it produced, Andrea says. “So it was not an additive thing. It was changed.”

From this mixture, Don discovered eight novel 16-membered macrolides. They named the first structure berkeleylactone A, which Andrea calls a “beautiful little compound” (J. Nat. Prod. 2017, DOI: 10.1021/acs.jnatprod.7b00133). The Stierles also isolated several known macrolides, including one discovered in the early 1970s by Eli Lilly and Company that the firm dubbed A26771B. When their colleague Jeremy Alverson tested the antibiotic activity of berkeleylactone A, he found it was extremely powerful against gram-positive bacteria, particularly those that were already resistant to other antibiotics, including methicillin-resistant Staphylococcus aureus (MRSA). Unlike other macrolides, berkeleylactone A doesn’t appear to kill bacteria by inhibiting protein synthesis or otherwise targeting the ribosome, giving it a novel mode of action.

“This was just incredible. It was something we had not expected to find,” Andrea says, although she’s still not clear on how and why this competition between fungi drives the production of this macrolide.

What the Stierles do know is that environmental conditions and natural selection can dramatically change an organism’s chemistry. Secondary metabolites produced by the microbes they pulled out of the Berkeley Pit looked nothing like those made by another organism of the same genus and species that they ordered from the American Type Culture Collection despite that the organisms were grown under identical conditions.

The Stierles’ painstaking comparisons are a case study for what is so challenging—and rewarding—about studying secondary metabolites, says Douglas Clark, a chemistry professor at the University of California, Berkeley.

“The pit environment is unusual in so many ways—it’s bound to produce different molecules with unique biological activities,” he says. “These environments have higher challenges, but they bring higher opportunities.”

For now, there are no shortcuts in studying an organism’s secondary metabolites. Scientists must still winnow the one-in-a-million chemical that might have pharmacological potential from all the compounds floating around extracts from cultured microbes. Then they must test it for biological activity and potential toxicity. Although scientists are developing sophisticated computer programs to glean more information about the precise composition and potential structure of these compounds from DNA sequences, the output currently remains relatively nonspecific, providing a broad category of compound rather than a specific chemical, Lodz University of Technology’s Boruta explains. When further work in computation and biochemistry can refine these approaches, the identification of a molecule’s structure from its genes will be a “game changer” in drug discovery, Boruta says.

Other microbiologists have been trying to eliminate the need to grow these organisms in the lab at all. Instead, they have begun to use metagenomics to flag genes of interest, make lots of copies with PCR, or synthesize the DNA from scratch. Then they insert the genes into easy-to-culture organisms like Escherichia coli and Saccharomyces cerevisiae and let those microbes crank out massive quantities of the secondary metabolites. The approach isn’t always successful, but it’s gaining traction, the University of Aberdeen’s Jaspars says.

After all this work, researchers still have to determine whether their promising molecule is active in human cells. The sheer effort required to do this research is why the Stierles have shifted their focus away from the anti-inflammatory compounds they’ve identified and toward antibiotics. Identifying whether a molecule has antibiotic activity is quicker and easier than determining if a given drug is killing cancer cells.

The Stierles attribute their success to a combination of hard work and good luck. Andrea’s lack of formal training in microbiology (“I’m not a microbiologist, even though I play one in the lab,” she laughs) has itself become an asset.

“You rush in where the better angels might fear to tread, and it can work out quite well,” she says.

She concludes: “It is amazing how this little pit lake, in the middle of a little town, in a sparsely populated state like Montana, can yield such richness. But that’s true of every ecosystem. They have secrets, and you’re lucky if you get to share some of those secrets. We’re recognizing that these little organisms are incredible chemists and can do a number of different things if we just know how to ask them.”

Carrie Arnold is a freelance science writer based in Richmond, Virginia.