Rapamycin’s secrets unearthed

On a snowy November day in 1964, a team of about 40 doctors and scientists boarded the Royal Canadian Navy’s H.M.C.S. Cape Scott in Halifax, Nova Scotia. They were headed to Easter Island, a triangle-shaped speck in the South Pacific that’s 2,200 km from its nearest inhabited neighbor. Their goal: to study a group of people—their heredity, environment, and common diseases—who lived in this uniquely remote spot before the Chilean government disrupted their isolation with an airstrip on the island’s southwestern corner. As the scientists set out, no one could have predicted that the Canadian expedition’s most valuable finding would come from a bit of bacterium ensconced in a sample of Easter Island’s soil.

Georges Nógrády wasn’t looking for cures when he divided Easter Island into 67 parcels and took a soil sample from each. The University of Montreal microbiologist was trying to understand why the islanders weren’t afflicted with tetanus, a bacterial infection often found in places with horses. Not only did horses outnumber the island’s inhabitants, but its people walked around barefoot—a notoriously easy way to pick up tetanus spores.

Nógrády ended up isolating tetanus spores in only one of his 67 samples. Those vials of dirt could have ended up as a footnote to a historical trip, were it not for scientists at Ayerst Pharmaceuticals. In 1969, the collection of soil samples was given to Ayerst’s Montreal research site, where a group was focused on natural products—specifically medicinal compounds made by bacteria.

It was a fateful exchange. Within one of those vials lived a bacterium with the ability to churn out rapamycin, a then-unknown macrocyclic compound that turns out to have myriad medicinal properties. The molecule was found to be a powerful immunosuppressant that can also stop cancer cells from dividing. Researchers have spent decades unraveling the form and function of the compound and its target, a protein known as mTOR. Now, nearly 50 years on from the Canadian expedition, rapamycin and related compounds, dubbed “rapalogs,” are finding new life as possible antiaging pills.

A rocky start

Finding and identifying novel compounds from Nógrády’s collection was a years-long undertaking. Ayerst scientists had to first isolate microorganisms from the soil, coax them to grow, and finally screen the brew of chemicals those microorganisms produced to see if it had any promising biological activity. One of the microbes, a bacterium identified as Streptomyces hygroscopicus, produced a compound that could kill fungi. After two years of work isolating that molecule and generating enough of it to deduce its structure, the Ayerst scientists found they had a new natural product. They called it rapamycin, after Rapa Nui, the name given to Easter Island by its indigenous people.

As the researchers were studying rapamycin’s antifungal activity, they discovered that the compound was not only a potent immunosuppressant, but also could keep cells from multiplying. Suren Sehgal, a microbiologist at Ayerst, sent a sample of the compound off to the U.S. National Cancer Institute for screening. NCI found that it had incredible activity against solid tumors.

“It was a totally new class of anticancer agents we were looking at,” said Sehgal in an interview with the Journal of the National Cancer Institute two years before his death in 2003. Up to that point, chemotherapies had all been cytotoxic—they killed living cells. Rapamycin was cytostatic: It kept cells from growing and dividing. Working together, NCI and Ayerst began to test rapamycin in combination with chemotherapeutic agents in mice with promising results.

But all work on rapamycin came to a sudden halt in 1982. That’s when Ayerst decided to shutter its Montreal research lab. The company laid off everyone but a core group of about 30 scientists who were moved to its facility in Princeton, N.J. With rapamycin proving difficult to formulate into an intravenous drug for clinical trials, the project was shut down.

“For all practical purposes, rapamycin was a lost cause,” Sehgal recalled in 2001. “It was abandoned by Ayerst as a drug candidate.” The remaining soil samples, isolates, and rapamycin were cataloged in Ayerst’s library and essentially forgotten by the company.

Sehgal did not forget about rapamycin, though. “He was convinced that this material was extremely valuable for saving human lives,” recalls Jehan Bagli, a friend of Sehgal’s and a chemist who worked on rapamycin at Ayerst in Montreal. Sehgal’s widow, Uma, remembers: “He used to say, ‘I have such an exciting product in my hands.’ ”

Before Ayerst shut down the Montreal site for good, Sehgal decided to do a large-scale fermentation of the rapamycin-making S. hygroscopicus. He packed the bacterium into some vials and took it home, where he stuck it in the freezer next to the ice cream in a package labeled “DON’T EAT!”

When the time came to move to Princeton, Sehgal’s son Ajai was summoned home from college to help. He was tasked with procuring dry ice to pack the freezer holding the precious bacterium. “I sealed the freezer with duct tape so that the movers wouldn’t open it,” he remembers. The bacterium stayed in the Sehgal family freezer for the next five years.

A new beginning

In 1987, American Home Products—the parent company of Ayerst—decided to merge Ayerst with Wyeth, another pharmaceutical firm it owned. Wyeth’s leadership took the helm of the new Wyeth-Ayerst Laboratories, and Sehgal felt he might find his new bosses more receptive to rapamycin research. He wrote a memo to top management in 1988 and was asked to give a presentation outlining what he knew about the compound. His new managers also told Sehgal to get in touch with outside investigators who could test the drug, which had immunosuppressant properties, in organ transplant animal models.

The time was ripe for developing drugs that suppressed the immune system. Sandoz’s cyclosporine had been approved to prevent organ rejection in transplant patients in 1983 and had turned out to be a blockbuster drug for the company. Around this time, researchers at Fujisawa Pharmaceutical Co. reported another immunosuppressant compound, FK-506. Half of its structure is identical to that of rapamycin.

FK-506 became the molecule du jour in the late 1980s and early 1990s as academic researchers sought the source of its biochemical action. The compound was approved by the U.S. Food & Drug Administration in 1994 to stave off organ rejection in liver transplants (and would later find use with other types of organ transplants) under the name tacrolimus.

Wyeth’s medicinal chemistry and pharmacology teams also took an interest in rapamycin, recalls Magid Abou-Gharbia, who spent 26 years with the company and is now director of the Moulder Center for Drug Discovery Research at Temple University. The chemistry team initially focused on formulating the compound so it could be given orally. That effort led to commercial success: Rapamycin was approved as an immunosuppressant to prevent organ transplant rejection in 1999. Pfizer, which bought Wyeth in 2009, markets it under the name Rapamune (sirolimus).

As rapamycin was wending its way through clinical trials for transplant patients in the mid-1990s, scientists at Wyeth turned their attention to using the compound to fight cancer. But they needed to modify the molecule, Abou-Gharbia explains, because the initial patent on rapamycin expired in 1992. The company had patents for the compound’s formulation and for its use as a transplant drug, but the company would need a novel compound to market it in oncology.

Working in collaboration with chemists at Columbia University, Abou-Gharbia’s team at Wyeth began to probe rapamycin’s protein-binding interactions. They wanted to figure out where they could make modifications to the molecule that wouldn’t alter its anticancer activity.

By this time, researchers in a few different labs had worked out that rapamycin actually binds two different proteins—called FKBP12 and mTOR—and brings them together. To avoid perturbing those interactions, the chemists identified a cyclohexane ring extending from rapamycin’s periphery. The ring had a hydroxyl group that could be altered to make novel compounds without any significant change to the binding of either FKBP12 or mTOR. This insight led to more than 100 patents, Abou-Gharbia says.

Wyeth eventually focused on an ester derivative of rapamycin as a cancer treatment. The resulting compound, Torisel (temsirolimus), was approved to treat kidney cancer in 2007.

Wyeth wasn’t the only company that zeroed in on rapamycin’s cyclohexane ring hydroxyl handle. Chemists at Novartis tacked a hydroxyethyl group onto that same part of the molecule to create their own rapalog. Their compound, Afinitor (everolimus), was approved to treat advanced kidney cancer in 2009. Approvals for other cancers and for use as an immunosuppressant to prevent rejection of transplanted organs followed. Other rapalogs, developed as cancer therapies or as compounds for drug-eluting stents, also capitalize on modifying that hydroxyl handle.

While medicinal chemists were tweaking rapamycin’s structure to make novel and improved compounds, other researchers were trying to understand the biological function and biochemical mechanism of its target, the aptly named mTOR, short for mechanistic target of rapamycin.

Scientists spent years figuring out that mTOR acts like a central hub for nutrient signaling. It kicks into gear based on the levels of amino acids, glucose, insulin, leptin, and oxygen in cells, explains Dudley Lamming, a professor at the University of Wisconsin who studies the physiological role of mTOR. “The point of integrating all these different environmental conditions is to determine when cells should grow,” Lamming says. That gauge is critical: If, for example, cell division is initiated without the right amounts of nutrients around to fuel the process, a cell would die rather than multiply.

Rapamycin works by blocking the activity of mTORC1, a distinct multiprotein mTOR complex that is responsible for coordinating all of that nutrient information. The natural product binds to another protein—FKBP12—and then latches with exquisitely selectively to a region on mTORC1 and effectively forms a blockade over its catalytic cleft. This, Lamming says, likely accounts for its immunosuppressant activity because it prevents the mTOR complex from coordinating the growth of B cells and T cells, the immune system’s sentries. Scientists think rapamycin uses the same mechanism to stop cancer cells from growing.

Fountain of youth?

By the mid-2000s, researchers had begun to study the effects of manipulating genes in the mTOR pathway. Within a two-year span, various research groups reported that dialing back mTOR’s activity in yeast, nematode worms, and fruit flies extended their life span. “These things all came out at about the same time,” says Matt Kaeberlein, a University of Washington Medical Center researcher who was a coauthor on the yeast studies. “None of us had talked to each other. Sometimes that happens in science.”

Kaeberlein says those studies of various organisms made it clear that mTOR was doing something fundamentally important. “Although they’re all simple organisms, they’re also very evolutionarily divergent,” Kaeberlein adds. “So it was clear that this was some fundamental aspect of aging that we’d tapped into.”

Because researchers already had a drug—rapamycin—that could turn down mTOR in the same way the genetic knockout did, they began to explore whether rapamycin could extend life span in model organisms. As part of the National Institute on Aging’s Interventions Testing Program, researchers in three different laboratories fed rapamycin to mice and studied how it altered their life spans.

“We’d planned to start rapamycin at eight months of age because we wanted the animals to fully finish growing” before dosing began, explains David Harrison, who was in charge of the mouse study at the Jackson Laboratory, one of the three sites. But the team had trouble finding a way to formulate the drug so it could be put into the mouse food. By the time the team had solved that problem, the mice were 20 months old. Still, the researchers decided to see what would happen if they gave rapamycin to this group of older mice.

The results, reported in 2009, made headlines. Male mice fed the rapamycin diet lived about an extra six months—9% longer than mice that didn’t consume the drug. Female mice did even better, living 14% longer than their counterparts in the control group. Experiments in younger mice that were published in the same report showed essentially the same extension of life span. “It didn’t really matter whether we started at nine months, which is equivalent to a person at age 35, or at 20 months, the equivalent of a person at 60 or 65,” Harrison notes.

The mouse study was part of a larger project to test many possible life-span-extending compounds in mice. The three laboratories each tested 25 compounds. “Of these, only six gave positive results, and most were only in males,” Harrison says. “Only rapamycin showed substantial benefits to both sexes.”

After the mouse study, scientists started to think about testing rapamycin and the rapalogs in other mammals, including people. “Most of the things that we know work in mice, to either correct disease or extend longevity, we have no idea if they will work in humans or other mammals,” says Adam Salmon, of the Barshop Institute for Longevity & Aging Studies.

Salmon and his collaborators are studying rapamycin in marmosets, tiny monkeys that typically live to about eight years old. Last year his team got funding to probe the effect of rapamycin on the marmoset’s longevity when given at middle age.

In Seattle, Kaeberlein’s group has been studying rapamycin in pet dogs as part of the Dog Aging Project. The researchers recently completed the first phase of the study, which included 24 older but otherwise healthy dogs that were given rapamycin or placebo for 10 weeks to determine the best dose of the drug that would have the fewest side effects. The study also looked at whether rapamycin has a positive effect on cardiac function in dogs, a benefit observed in mice given the compound. It seems to, but Kaeberlein is reluctant to draw too many conclusions from such a small study. Even so, he’s encouraged and plans to scale the study up to examine rapamycin’s effects in more dogs over a period of several years and to look broadly at parameters of aging, such as cognitive function, cancer rates, and kidney function.

But longevity researchers are most excited about the effect inhibiting mTOR has in humans. In 2014, Novartis reported results from a trial in which 200 elderly people took a pill containing either placebo or one of three doses of the rapalog everolimus over the course of six weeks. Then, after a two-week break, the firm’s researchers gave all the study participants the flu vaccine and looked at their immune response. Subjects on the lowest dose of everolimus seemed to do the best, as measured by the presence of flu antibodies in their blood .

“There’s this extensive body of literature showing that in every species studied to date—yeast, worms, flies, and mice—if you inhibit mTOR, you extend life span and ameliorate a variety of aging-related diseases,” says Novartis’s Joan Mannick, who led the study. “We thought it wasn’t such a leap of faith to think that inhibiting mTOR in humans might have some beneficial effects on aging-related conditions.” The tricky part, she says, is most aging-related conditions in humans occur over many, many years, making it tough to do clinical studies. Because immune function declines with age and can be tested in a relatively short time frame, it seemed ideal as a marker for studying the effects of mTOR inhibition in people.

It might seem contradictory that everolimus, an immunosuppressant, can boost immune function. But Mannick says that there are a couple of reasons this might be so. Transplant and oncology patients generally take high doses of mTOR inhibitors, which essentially turn the pathway off. In Mannick’s study, patients took low doses of everolimus, which turned the pathway down. “There are preclinical data that show as you age your mTOR is overactive, so you just may need to turn it down to ‘young’ levels to help some organs, including the immune system, work better,” Mannick says.

Next questions

Rapamycin and the rapalogs aren’t the only things that inhibit the mTOR pathway. Calorie restriction, which is also known to increase life span, does the same thing. It makes sense that calorie restriction, which limits nutrients, shuts down mTOR. But why does inhibiting this pathway have an effect on aging?

We don’t exactly know, says David Sabatini, a scientist at the Whitehead Institute who, as a graduate student, was among the first to discover mTOR in the mid-1990s. He has been studying the protein ever since. “Lots of things go wrong with aging, and the only way you can actually impact lots of those things is by affecting a pathway that’s really a master regulator,” Sabatini points out. We’re unlikely to find just one thing that mTOR does in cells to impact aging, he adds. “The reason that inhibition of mTOR works as well as it does is because it impacts many things.”

But don’t rush out for a rapamycin prescription just yet. The scientists who spoke with C&EN say it’s too soon to tell if mTOR inhibitors are safe for long-term use, something that would be required of an antiaging treatment. Given the option, most said they wouldn’t take it.

“I think it’s not the efficacy of rapamycin that’s in question. It’s the toxicity that everybody is concerned about,” says Brian Kennedy, president and chief executive officer of the Buck Institute for Research on Aging. Rapamycin and the approved rapalogs have side effects, such as mouth sores, but the main concern is their ability to suppress the immune system. The possibility of an extended life span with rapamycin might be cut short by an infection.

Now several researchers, including Kennedy, are trying to develop compounds that have rapamycin’s ability to inhibit mTOR without suppressing the immune system. “The cool thing about rapamycin is that nature spent millions of years perfecting this molecule,” Kennedy says. “It’s not optimized for what we want—which is treating disease or slowing aging—but it’s pretty darn good at what it does, and if we can tweak it in ways that make it better, I think there’s a really exciting opportunity.”