Albert Lasker
Clinical Medical Research Award

Award Presentation by Joseph Goldstein

One hundred years ago, two noteworthy events took place in Stockholm, Sweden. Both had far-reaching effects on the biomedical sciences. One was the settlement of Alfred Nobel's controversial will and the creation of the Nobel Foundation. This event had immediate impact and is well known to all of us. The second Stockholm event, occurring at precisely the same time, involved a remarkable series of experiments that laid the groundwork for this year's Lasker Award in Clinical Research.

In 1896, two physiologists at the Karolinska Institute, Robert Tigerstedt and Per Bergmann, injected extracts of kidneys into the bloodstream of rabbits, and they made a dramatic observation: The extracts produced an acute elevation in the blood pressure. These observations led Tigerstedt and Bergmann to propose that the kidney secretes a hormone with vasopressor properties. They named this hormone renin, befitting its renal origin, and they advanced the concept that renin could form the link between kidney disease and hypertension. The Swedish scientists published their results in 1898 in the Scandinavian Archives of Physiology. But unlike Alfred Nobel's one-page will published in the same year, Tigerstedt and Bergmann's 48-page article had no impact. Like Nobel, it lay buried in Sweden for the next 36 years.

Then, in 1934, Harry Goldblatt, a Cleveland physician, placed a clamp on the artery leading to the kidney of a dog and produced the first animal model of chronic hypertension. Goldblatt proposed that the decrease in blood supply causes the kidney to release a vasopressor substance. Goldblatt was unaware of the earlier discovery of renin. The connection between the Goldblatt kidney and renin was not made until the 1950s when scientists delineated the renin-angiotensin system, which is the major mechanism the body uses to control blood pressure.

When blood volume falls, the kidney releases renin into the bloodstream. Renin is not a true hormone; instead it is a proteolytic enzyme that starts a proteolytic cascade. Renin cleaves a large plasma protein to liberate a small 10-amino acid peptide called angiotensin I. Angiotensin I also does not directly raise the blood pressure. It must first be cleaved by another protease—the angiotensin converting enzyme. Angiotensin converting enzyme, affectionately known as ACE, clips two amino acids from the carboxy-terminal end of angiotensin I to produce an active 8-amino acid peptide called angiotensin II. Angiotensin II is the most potent natural vasopressor made by the body. It raises blood pressure by two actions. First, it constricts blood vessels, narrowing their diameter and raising their resistance. And second, it triggers the release of a steroid hormone called aldosterone, which induces the kidney to retain salt and water, thereby over-filling the circulatory system. In 1958, the Lasker Award in Basic Research was given to Irvine Page of the Cleveland Clinic for his pioneering research in delineating the renin-angiotensin system.

Under ordinary circumstances, the renin-angiotensin system is essential for health. It allows the body to maintain a stable blood pressure under conditions in which blood volume is depleted, such as after vomiting, diarrhea, or vigorous exercise. But in certain individuals, the renin-angiotensin system becomes overactive. It raises blood pressure inappropriately. The high blood pressure damages blood vessels, leading to strokes, heart attacks, heart failure, and kidney failure. Inhibitors of the renin-angiotensin system should be ideal agents to lower blood pressure and prevent the complications of hypertension, but how can we block this complex system? This problem was solved by David Cushman and Miguel Ondetti, the recipients of this year's Lasker Award in Clinical Research.

The story behind their discovery is a fascinating one, full of twists and turns. It begins in the banana plantations of southwestern Brazil where workers in the field would suddenly collapse after being bitten by a pit viper snake called Bothrops jararaca. This collapse was due to a sudden and catastrophic drop in blood pressure. In the late 1960s, scientists working in London in the laboratory of Sir John Vane discovered the reason for the sudden drop in blood pressure: The snake venom contained potent peptide toxins that inhibit ACE, the enzyme that generates angiotensin II. News of this discovery spread around the world, and the race was on to find a safe and orally active ACE inhibitor that would lower blood pressure.

At the Squibb Institute for Medical Research, now Bristol-Myers Squibb, two young scientists—David Cushman, an enzymologist, and Miguel Ondetti, a protein chemist—were assigned to investigate this problem. In 1970, their first step was to purify the molecule in the snake venom that inhibited ACE and then determine its chemical nature. The most potent ACE inhibitor in venom turned out to be a peptide that contained only nine amino acids. The Squibb scientists synthesized this peptide in pure form and provided it to two clinical investigators, Hans Brunner and John Laragh, then at Columbia P&S Medical Center. Brunner and Laragh administered the 9-amino acid ACE inhibitor to hypertensive patients and showed that it was extremely effective in lowering blood pressure. The therapeutic principle was established, but there was one serious problem. The purified peptide did not work orally; the peptide was too large to be absorbed from the gut. It could only work when given by intravenous injection, ruling out its use for the chronic treatment of hypertension.

Cushman and Ondetti set about to convert the 9-amino acid peptide into an oral peptidomimetic drug. Traditional chemical approaches did not work. In the meantime, scientists at Squibb began to screen thousands of chemicals that had been synthesized for other purposes. None of these "off-the-shelf" chemicals inhibited ACE; the mass screening effort was a failure, and the ACE project was terminated. Cushman was shifted to a program in antibiotics and Ondetti to prostaglandins.

But the curious Cushman and the unstoppable Ondetti never lost their fascination with ACE. They continued to discuss ways to develop an oral inhibitor based on the sequence of the snake venom peptide. They had several ACEs up their sleeves. First, they were struck by the resemblance of ACE to a well-studied digestive enzyme called carboxypeptidase A whose atomic structure had been determined by X-ray crystallography several years earlier by William Lipscomb at Harvard. Like carboxypeptidase, ACE cleaves amino acids from the carboxy-terminal end of peptide substrates. Like carboxypeptidase, ACE contains a tightly bound zinc atom in the active site. But there was one fundamental difference between ACE and carboxypeptidase. ACE split off the last two amino acids from the end of its peptide substrate, whereas carboxypeptidase split off only one.

From Lipscomb's atomic structure, Cushman and Ondetti knew how carboxypeptidase split off the last amino acid from the peptide substrate. The substrate bound to the enzyme at a site called the peptide binding site. Immediately next to this site was the zinc atom that interacted with the peptide bond that was to be cleaved. Cushman and Ondetti knew one other crucial fact about carboxypeptidase. This fact they learned on the afternoon of Wednesday, March 13, 1974, when they happened on a year-old research article by Byers and Wolfensen that described a potent inhibitor of carboxypeptidase. This was the epiphanous moment in the discovery of the ACE inhibitors.

The inhibitor that Cushman and Ondetti read about was designed to bind tightly to carboxypeptidase, blocking entry of the normal peptide substrate. As soon as they discussed this paper, Cushman and Ondetti perceived something that other scientists had overlooked: the carboxypeptidase inhibitor worked so well because it contained two functional groups in the same molecule—one of its functional groups mimicked the phenylalanine amino acid to be cleaved, and it bound to the peptide binding site. The other functional group, called the succinyl component, bound to the adjacent zinc atom. That a single inhibitor molecule would bind to two different sites in the same enzyme was quite unusual. Cushman and Ondetti leapt into action: They created a model for the perfect ACE inhibitor based on the carboxypeptidase model.

As their first candidate ACE inhibitor, Cushman and Ondetti chose a modified version of the carboxypeptidase inhibitor in which they changed the phenylalanine amino acid to a proline to mimic the last amino acid in the snake venom peptide that inhibited ACE. This compound showed slight positive activity in their biological assays, indicating that they were on the right track. The breakthrough came 60 compounds later when they replaced the succinyl group with a derivative of cysteine. The sulfhydryl of the cysteine bound zinc much more tightly than the carboxyl of succinyl. Compared with their first compound, the cysteine-proline inhibitor was 30,000-fold more potent. Now remember, Cushman and Ondetti were making all of these molecular manipulations without the benefit of an atomic structure of ACE itself; they were relying on inferences deduced from the X-ray structure of the related carboxypeptidase.

In a final tuning of their cysteine-proline inhibitor, Cushman and Ondetti introduced a methyl group that rendered the drug resistant to attack by peptidases in the stomach and bloodstream. The result was the drug that we now know as captopril—the first orally active ACE inhibitor. As Mae West would say, "The rest is history."

From the moment of conception of their model on March 13, 1974, Cushman and Ondetti took only one and a half years and only 60 synthetic modifications to create captopril. Captopril is a truly amazing achievement: It is a derivative of only two amino acids—one of the simplest, yet one of the most optimized of any drug ever taken by patients. This was the first time that scientists exploited a three-dimensional protein structure to design a drug, ushering in a new technology called "structure-based drug design," which is now used throughout the pharmaceutical industry. The HIV protease inhibitors for AIDS patients were developed via a structure-based approach similar to that pioneered by Cushman and Ondetti. Five new drugs, developed by structure-based design, are currently in clinical trials for treating glaucoma, cancer, psoriasis, thrombosis, and the common cold.

Captopril was approved by the FDA in 1982 for the treatment of hypertension. One year later in 1983, it became the first new drug in 15 years to be approved for the treatment of congestive heart failure. In 1994, captopril won FDA approval as the first drug that prevents kidney disease in patients with diabetes mellitus. In addition to captopril, nine different ACE inhibitors have now been approved for use in patients in the U.S. Today, ACE inhibitors constitute the major class of drugs used for the treatment of hypertension and its complications.

Hypertension is one of the most common diseases in our society. Fifty million Americans, one of every four adults, have high blood pressure. Antihypertensive drugs are the most frequently prescribed medicines in the U.S. Hypertension is not only common; it is also deadly. Its presence accelerates the atherosclerotic process, producing strokes, heart attacks, heart failure, and kidney disease. Each year hypertension costs this country more than $8 billion in health care expenses and lost productivity. Last year alone, hypertension was responsible for 500,000 deaths in the U.S. More than half of these deaths occurred in people who either never knew they had hypertension or were never treated effectively.

It's hard to believe that one of these people was the most powerful statesman in the world. Franklin D. Roosevelt was first diagnosed in 1937 with severe hypertension. He was treated with the most potent antihypertensive therapy at the time—bed rest, salt restriction, and phenobarbital. Not surprisingly, his blood pressure did not drop. In 1940, FDR suffered the first of several bouts of heart failure, which were treated with digitalis. His blood pressure continued to rise and at the Yalta Conference in February 1945, he was extremely ill with blood pressures in the range of 250/150. Two months later, FDR died suddenly from a massive cerebral hemorrhage. If ACE inhibitors had been available 40 years earlier, world history might surely have changed.

The good news is that the dread consequences of chronic hypertension are becoming less frequent as more and more people are diagnosed. If hypertension is detected in the early stages, it can now be effectively and safely treated—thanks to this year's Lasker Clinical Awardees, David Cushman and Miguel Ondetti. You've all heard about the "ace in the hole." Well, Cushman and Ondetti discovered the "hole in the ACE" and plugged it with their inhibitor!