by Rob Dunn
Fleming had discovered penicillin, but also, in the process, the ancient wars between bacteria and fungi. Nearly all fungi that have been studied in detail now appear to produce at least some compounds that they use to kill bacteria (fungal antibiotics still account for 65 percent of those on the market). If there are one million fungal species (no one knows even roughly how many there might be, but this seems a reasonable guess), there are, buried in their bodies and genes, as many kinds of antibiotics, antifungals, and other useful drugs. We need antibiotics; we thrive thanks to them. But Endo had a broader insight, one that no one else seemed to have had—that one could use the antibiotics produced by fungi for ends entirely unrelated to killing bacteria. To get started, all one had to do was think about how fungi might attack bacteria and consider how that attack might be put to use.
Among the simplest ways for a fungus to destroy bacteria is to attack their cell walls (as with penicillin) and, inside those walls, their cell membranes. Cell walls hold bacteria together and keep them from diffusing into oblivion. Cell membranes serve as permeable filters. Endo knew that the cell membranes of many bacteria were built on a scaffolding that included compounds similar to cholesterol. What if, he speculated on these plausible evolutionary grounds, some fungi had evolved ways to stop the production of the cholesterol bacteria required in their cell membranes?
Endo would search for such a fungus. The idea that it might exist was reasonable—at least with hindsight—but the possibility that he would find it was far-fetched. He was searching for a compound he had speculated might exist, but he didn’t have any real guess as to which species it might be in. He decided to study a few thousand species of fungus. If he needed to, he would study more. He would spend years, if necessary, even decades. The only real limit was how long his employer would give him to search and, of course, how many kinds of fungi he could find to test. The task required many steps. Each fungus had to be found in nature and then grown in broth in giant flasks (which required knowing what to feed it). The flasks would then be stirred up with the liver enzymes (from rats) necessary to produce cholesterol.6 Fungi able to stop the enzymes would then be studied some more. Among those, unsafe fungi would be discarded. Expensive fungi would be discarded. Fungi that were slow to reproduce would be discarded. Fungi that broke down when heated would be discarded. Then, when he had a list of fungi that passed all these tests, Endo would have to do even more tests to figure out what was going on, why the fungus seemed to work. Endo would double-check whether the rate-limiting enzyme animals use to produce cholesterol, HMG-CoA reductase, was inhibited. He would then try to isolate and purify the active compounds that were doing the inhibiting.
The work was tedious and grueling, less science than factory work, industrial microbiology. As Endo would say in one interview, it was “unlike a lottery, which must include prizes… [In the search for drugs] no one knows if there is a prize.” Each hopeful day was followed by a dozen hopeless ones. But finally, after screening thirty-eight hundred fungal species, a potentially useful antibiotic, citrinin, was found in the fungus Pythium ultimum. When injected into the bloodstreams of rats, citrinin inhibited HMG-CoA reductase and, more important, lowered cholesterol levels. There was a moment of ecstatic excitement in the lab, but then citrinin proved toxic to the kidneys of the rats. Back to the drawing board.
Then, one day in the summer of 1972, after another twenty-three hundred compounds had been tested, another compound was found that appeared to reduce the function of HMG-CoA reductase and, hence, cholesterol production. Once again, Endo and the lab were excited. Once again, they confronted a challenge. They couldn’t isolate the compound in the fungal strain that was having the effect. Finally, in July of 1973, a breakthrough: they found the magical compound later named compactin. Compactin’s mechanism of action was simple. It resembled the normal substrate to which HMG-CoA reductase binds, and this allowed the compactin to gum up the HMG-CoA reductase and prevent the production of cholesterol. In nature, this would allow the fungi to fight bacteria by preventing them from building cell walls. But what Endo hoped is that it might also allow him to fight heart disease by preventing cholesterol from becoming so abundant in the blood that it led to plaques.
The fungus from which Endo isolated compactin was found growing on rice in a grain shop in Kyoto; it also grows on fruits, including oranges and lemons. Compactin seemed, from the start, safer than citrinin. Maybe it was the compound he had been working toward. What was even more amazing to Endo was that the compound was produced by Penicillium citrinum, close kin to the fungus that had helped Fleming discover antibiotics in the first place. This alone seemed auspicious, but the key test was whether the compound could lower cholesterol levels in rats. Endo sent the compound to be tested at the Central Research Laboratories of Sankyo. He could do nothing but wait. Then the test came back. The cholesterol levels in the rats had not dropped. Endo’s mind filled with despair and expletives. He tried again, this time performing the test himself on both rats and mice. Once again, no luck. As he would later say, “It looked as if two years of work and over six thousand tests had led nowhere.”
At this point, many people would have given up. Giving up would have been forgivable; it would have been reasonable. Some discoveries are inevitable; the discovery of statins, the drugs that Endo would ultimately prove to be searching for and of which compactin was one form, was not necessarily one of them. But Endo did not give up; he picked himself up out of his exhaustion and tried to figure out what had gone wrong with the rats. Slowly, progress was made, and then, two years later—seven years after the idea for this project had originally occurred to him—he remembered the chickens! They were to be, he imagined, the chickens of hope. He had a friend, Noritoshi Kitano, with chickens. Perhaps, he thought, something about the rats he was using was unusual. The rats, he knew, had less cholesterol in their blood than humans did. Maybe the rats were weird. It was an extraordinarily remote possibility—after all, rats are the model for humans used in labs around the world, and the reason for using these model organisms is their similarity to humans. Humans and chickens are, well, different. But it was a possibility all the same, and the last unchecked Lotto ticket in a lottery that might not have a winner.
Kitano was about to kill his chickens, but over drinks at a bar Endo begged him not to. Endo knew how to take care of chickens. He soon scooped up his friend’s chickens and spent the next weeks feeding hens and shoveling chicken shit while he tested them, one by one, and gave some of the birds his compound. On the thirtieth day, he went to check the chickens. He drew their blood. As he did, he wondered what on earth he was doing. If the compound did not work in rats, there was no reason it should work in chickens. But maybe, just maybe… Chickens do have high levels of cholesterol in their blood, much of which, at least in hens, ends up in their eggs. (Rats, by contrast, although Endo did not know it, have very, very little LDL cholesterol, the form that inhibits HMG-CoA reductase.) The chickens that had not been given the new compound had levels of cholesterol similar to those they started with. This was what had been expected. But what about those that had been given the compound? They would be the big test. What about them? In the experimental chickens, cholesterol levels had dropped by half! Half! Endo had just solved the cholesterol problem with a dozen chickens too old to eat. He would go on to repeat his experiment with monkeys, which were much more similar to humans than rats or chickens were. In the monkeys, the compound worked too.
The next steps (including testing compactin in dogs and in more monkeys) would prove challenging too, but in the end, Endo had achieved his dream of a chemical key to lock and close the body’s cholesterol doors. What was left was going commercial. After all Endo had been through, commercial challenges might have seemed trivial. They were not. First, there was a residual worry: Was this drug safe? One study in rats (in which compactin didn’t even work) suggested the drug made crystals form in the kidneys. It took Endo a year to show it did not, but Endo’s company i
nsisted that the compound did. They then argued (using data that have still never been shared) that compactin caused tumors in dogs. Endo’s project was discontinued. Endo appealed the decision and tried every other route he could, but to no avail. It was over; all of his work was for naught, at least for Endo.
Endo’s work on compactin encouraged others to search for statins, in some cases clandestinely. The drug company Merck had obtained a sample of mevastatin and unpublished data about it in 1976 as part of a disclosure agreement. Merck would not be so bold as to sell mevastatin, Sankyo’s drug, Endo’s drug, but it was sufficiently bold to study mevastatin and then look for similar drugs. Lovastatin (Mevacor) was isolated from the fungus Monascus ruber and then also another fungus, Aspergillus terreus. Lovastatin was approved by the FDA in the fall of 1987 and became the first statin drug marketed in the United States. Later, synthetic statins were produced by chemical analogy with the biologically produced versions or by simply altering the natural versions, synthetic statins that have led to Lipitor and Crestor, among other drugs, which are now collectively the most profitable type of drug on the market. As for Endo, he never benefited financially from his discovery. Endo left Sankyo, unceremoniously, in 1978. He took a job at the Tokyo University of Agriculture and Technology, where he has continued his research ever since with modest funding and no direct benefits from his statin research. As for his own cholesterol, he took Mevacor, Merck’s drug, for a while but then stopped, choosing to exercise more, he says, instead. Meanwhile, the statins themselves have withstood their subsequent tests. Tens of billions of dollars of statins are now sold globally each year. More than thirty million people are now taking statins, and, as a result, tens of millions of additional years of lives have been lived.
That statins save lives, particularly in older people, whose arteries are more likely to have begun to clog, is unequivocal. Where they have come under debate is in terms of just how universally they should be used (whether they should be used in the young, for instance, or in those with other medical conditions). In addition, recent research has suggested that their benefits might be due to both reductions in cholesterol levels and an anti-inflammatory or even antioxidant effect. Yet, in the complex story of the heart, statins are about as close as one gets to an unambiguously beneficial treatment. They were made possible because Endo was willing to spend thirty years of his life thinking like a fungus, thinking evolutionarily. Between Fleming and Endo and a solitary genus of fungus, a single experiment has already contributed more to the survival of humans than all medical technology combined. How much more do the other three hundred species of Penicillium have to offer? No one has fully checked. No one has even yet named most of the species of Penicillium, much less studied them. In my lab alone, we have found dozens of new species of Penicillium fungus living on and in houses, and tens of thousands of unnamed species of fungi more generally. How much more magic do these and the other hundreds of thousands of unnamed and unstudied fungal species have? The amount is large but unknown. Since 1971, the study of wild animals, plants, and, especially, fungi and bacteria has yielded 70 percent of all discoveries of new, medically useful compounds. These include antibiotics, antifungals (fungi must fight other fungi too), and even cancer treatments, in addition to statin drugs. But most species on Earth, tens of millions of species, have not yet even been named, much less studied. Medically important discoveries wait in each scoop of soil; they grow on each piece of moldy bread and in every rotting leaf or log, anywhere ancient lineages wage war or coordinate peace with chemicals, anyplace where anything is alive.7
12
The Perfect Diet
Akira Endo sought to lower the cholesterol levels in the body through evolutionary chemistry. Though one can debate how often the drugs Endo discovered should be used, it is clear his insights yielded sweeping consequences. Many of us will live longer thanks to Endo’s research. But from the very beginning of cholesterol’s story there has also been another approach to lowering it, one that many view as more natural—diet. The story of heart disease and diet is towered over by one figure, a kind of complicated forefather who, depending on your perspective, is either the hero or the villain of our modern dinner tables: Ancel Keys.
Keys was born in 1904 in Colorado Springs, Colorado. When he was a small boy his family moved to what they hoped would be the prosperity of San Francisco to look for jobs; then the great earthquake split the ground open. The family moved again, this time around the bay to Berkeley, California. Even as a child, Keys seemed marked for some form of greatness. A study of child geniuses labeled him as one of the true young intellects. Perhaps because of his intellect, he was restless. He wanted to explore the world on his own terms, leaving for adventures even before he had graduated high school. He worked in a lumber camp, shoveled bat guano, and traveled to Asia on a ship on which he worked as a mechanic. This wanderlust was punctuated by the moments of stability during which Keys would achieve important milestones. Between trips, he started and finished an undergraduate degree. During a later period, beginning in 1927, he enrolled in the PhD program at the Scripps Institution of Oceanography. There, he began to study fish. Among their feathery gills and puckered mouths, the flower of his real genius began to unfold, a genius for big scientific projects, adventures in the unknown. It was this mature genius that would eventually turn toward the study of human bodies, diets, and hearts.
But not at first; he still had to figure out the fish. At Scripps, Keys focused on physiology and how fish—especially killifish—dealt with hypoxia, the absence of oxygen, an absence felt first in the active muscle of their hearts. It was in studying hypoxia that he became interested in the body’s powerful ability to regulate itself, its tendency toward homeostasis. After finishing his PhD, during a brief stay in Copenhagen with the Nobel laureate August Krogh, he built on his work with killifish to understand how eels could keep the salt concentration in their blood the same as they moved from salt water to freshwater and back. Keys readily made new discoveries, the ease and frequency of which inspired in him a complicated mix of humility (at the grandeur of what we don’t know) and boldness. Had he stopped all research right there, he would still be remembered, but he did not stop.
In 1933, Keys returned to the United States and decided to try his hand at studying humans as part of the Harvard Fatigue Lab. How different, after all, could humans be from fish? Keys was to be part of a growing number of scientists who had become interested in the ability of the human body to maintain its normal function under difficult conditions (much as fish do in different bodies of water). Keys was particularly interested in studying the body’s response to high elevations. Toward this end, he led an expedition to observe the acclimation of bodies (including his own) to high elevations, an expedition that allowed him to simultaneously satisfy his wanderlust and advance science. The expedition, in which Keys led a large group of scientists, was a collective self-experiment, something very common for exercise physiologists, particularly those at Harvard. The scientists would measure how their own bodies responded to high elevations. Predictably, the journey was not without challenges (including a high-elevation fistfight), but it was a scientific success.1 On his return, Keys wrote articles about the size of athletes’ hearts (much larger than normal), the properties of insulin, the chemistry of blood, the exchange of carbon dioxide between mother and fetus in goats, the permeability of capillaries, the ways in which oxygen moves from blood into tissues, the effects of testosterone and estrogen on the body, and much more. The papers were insightful, bold, creative, and, like him, wandering. For this work, he was lauded and rewarded. The largest of the rewards was (after his time at the Mayo Clinic, where he met his wife, Margaret), the establishment, in 1937, of his own lab, the cryptically named Laboratory of Physiological Hygiene at the University of Minnesota.
All of the studies Keys did, at his own lab and elsewhere, would help him in his ultimate quarry, the study of human hearts and cardiovascular disease. The high-elevati
on study taught him how to lead lab scientists into the wilds of the field, for example. But perhaps the most useful (and foreshadowing) experience Keys had was the challenge of developing a perfect military food. The military was unhappy with the food that paratroopers were getting. Keys ran a physiology lab. He thought himself sufficiently clever and experienced to do the job, and so he went to the Quartermaster Food and Container Institute for the Armed Forces, in Chicago, and suggested to those in charge that he could solve their problems. They declined his help, but he was able to get support from the Cracker Jack Company in the form of Cracker Jack boxes and a small amount of money from William Wrigley’s office (the same Wrigley of spearmint-gum fame). With the Wrigley funds, Keys bought things from the local supermarket and tried to produce a meal that, in light of his other studies on vitamins and energy requirements, was both balanced enough to be sustaining (given the knowledge at the time) and tasty enough to prevent revolt. He wanted to make the food healthy. He was not yet thinking about the health of the heart (though that would come), but he already knew that success with diets needed to be equal parts taste, marketing, and science. The approach worked. Keys produced what would later be called the K ration, a package of food items intended to be used only by paratroopers in emergencies. But military leaders liked it so much (General Patton thought it a military breakthrough) that it became the standard ration given to all fighting men. It was so successful it grew to be normal.
