A lifetime making mischief with DNA

As HIV prepares to replicate in fresh cells, it produces a long strand of polyprotein. To complete the process, this strand then breaks apart into smaller units made up of essential viral proteins and enzymes. In a circular bit of logic typical of the virus, it is one of these essential enzymes — protease — that starts this process rolling by separating the polyprotein strands in the first place.

Protease inhibitors have kept HIV at bay in many patients by limiting its ability to reproduce infectious virus. William Prusoff believes he has found a new way to keep the enzyme from triggering that protein fragmentation. In a laboratory freezer next to his office in Sterling Hall of Medicine, he keeps a supply of HIV protease stored at minus 80 degrees centigrade. Vials of boron analogs of selected tetrapeptides are close at hand. By chemically inserting the boron into polypeptides that interact with the protease, it is possible, he theorizes, to shut down the replication of HIV. “If one inhibits the cleavage of the polyproteins, a virus is made, but it is not infectious,” he says, sitting in his office in the Department of Pharmacology. “We feel that it will be a very potent inhibitor.” Although Prusoff’s boronated peptides have shown promise in laboratory experiments, he knows it will take years to determine if they are effective in humans.

At 79, Prusoff is used to this painstaking pace. He has spent his career making mischief with DNA. Four decades ago, his work led to the first antiviral drug, a compound used in the treatment of herpes infection of the eye. In the 1980s, he and his late colleague Tai-Shun Lin, Ph.D., made the discoveries that enabled development of d4T, a reverse transcriptase inhibitor marketed by Bristol-Myers Squibb under the trade name Zerit. In the year ending June 30, sales of Zerit generated $40 million in royalties for Yale, nearly all its income from the licensing of intellectual property, and extended the lives of tens of thousands of patients with HIV. (Total sales were $551 million, based on 76,755 prescriptions.) These successes have brought Prusoff, a senior researcher and professor emeritus of pharmacology, the respect of basic scientists and physicians around the world and general acknowledgement as the father of antiviral chemotherapy.

Prusoff wears this crown lightly, almost reluctantly. He is glad to talk about his work and, if pressed, will admit to developing the first antiviral. “What it showed was that you could get a compound which would be effective clinically in a viral infection,” he says with enthusiasm. But call him a trailblazer and he takes a step back. “I look at my career as one of serendipity,” he says. This decidedly low-profile approach is typical of the man, his friends say. “Bill Prusoff is a very humble man, a real gentleman,” says Alan C. Sartorelli, Ph.D., Alfred Gilman Professor of Pharmacology and Epidemiology and interim chair of pharmacology. “There are faculty around the country, at Yale even, who have this air of superiority. Not Bill Prusoff.”

Although retired, Prusoff continues to probe the complexities of thymidine, the molecule that has been his lifelong interest. One of four nucleosides in DNA, it has been the source of his major accomplishments since the 1950s, when he struck upon the therapeutic value of idoxuridine, which became the first antiviral compound approved by the FDA. His discovery gave physicians their first weapon against herpes and the related keratitis, then the leading infectious cause of blindness. Idoxuridine is a thymidine analog. Once incorporated into the viral DNA, it leads to the synthesis of abnormal RNA and proteins. “It stops the infection because the virus can’t reproduce properly,” Prusoff says. “People were looking at antivirals before then. This was the first one which was successful.”

With this compound, Prusoff did more than invent a new drug. He turned the prevailing wisdom about antiviral drugs on its head. According to David Barry, M.D. ’69, HS ’72, founder of the drug-discovery company Triangle Pharmaceuticals Inc., the dogma was simple: Antivirals could not work. If effective, they would prove too toxic for use in humans. If safe for human use, they would lack the potency to control the virus.

“What Bill showed was that that was not always the case,” says Barry, who studied antiviral compounds in Yale’s pharmacology department, though not with Prusoff. “That major leap of thought is really what started antiviral chemotherapy.”

“He showed that you could control or obliterate viral infection by interfering with the nucleic acid processing,” says Gregory Gardiner, Ph.D., former director of the University’s Office of Cooperative Research. “Every antiviral drug since then has worked that way until protease inhibitors came along for AIDS.”

Much remained to be done to develop antivirals, however. Although effective topically, idoxuridine was too toxic for systemic use, Prusoff says. Twenty years later he developed an amino analog of idoxuridine for systemic use against herpes simplex. It inhibited the virus, but left uninfected cells alone. “He was the first to demonstrate the feasibility of developing nucleoside analogs as a selective anti-herpes-virus compound,” says Yung-Chi Cheng, Ph.D., Henry Bronson Professor of Pharmacology, a long-time colleague and friend. Prusoff’s compound, however, had limited potency and was soon eclipsed by acyclovir, an antiviral nucleoside analog with a similar selective mechanism that proved even more effective.

In the 1980s, when Prusoff and colleague Tai-Shun Lin were looking for nucleosides that inhibited HIV, they turned again to thymidine. “It had been shown,” Prusoff recalled, “that a number of nucleosides had activity against the AIDS virus.” Thymidine by itself had little effect on HIV, but Prusoff and Lin tried a different approach. “People had not looked at thymidine with an unsaturated bond,” Prusoff said. “Dr. Lin and I decided to look at this compound, which had been synthesized in the 1960s by Jerome Horwitz at the Detroit Cancer Center. It had limited anticancer activity. We examined it for its effect against HIV and found it was very, very active.” As it became incorporated into the viral DNA, d4T shut off the virus’s reproductive mechanism. In 1992 d4T became the first drug tested under the FDA’s parallel track policy, which gave people with life-threatening illnesses access to drugs still in clinical trials. Zerit received the FDA’s approval in 1994. “It is clearly one of the most significant AIDS medications and one that has lasted in terms of market share,” says Barry, who led the team that developed AZT, the first medication to slow the progress of HIV.

Prusoff’s interest in chemistry started in high school. “It gave you an understanding of what the world was made of,” he says. “It fascinated me to learn that a piece of wood is made up of carbohydrates and a lot of atoms.” In 1937 he left his home in Miami to study chemistry at the University of Florida at Gainesville. “During the Depression, when I wanted to quit school and help out, my parents absolutely refused and insisted that I stay in school,” he says. His mother and father, Russian immigrants who had moved from New York City when he was 15, ran a small grocery store. “They had absolutely no education at all, yet they realized the importance of an education.” After a year in Gainesville, Prusoff transferred to the University of Miami so he could live at home and save the expense of room and board. He covered his tuition by taking a job as an assistant in the chemistry lab. In typical Prusoff style, he says his older brother was the smarter of the two. “He majored in sororities and minored in fraternities and became a very successful salesman,” Prusoff says fondly.

Following his parents’ wishes, Prusoff applied to medical schools, including Yale. He was turned down. “I was disappointed,” he says, “but it was probably the best thing that happened to me. I think I am a much better scientist than I would be a physician.” World War II put his graduate studies on hold. His poor eyesight kept him out of the Army while his chemistry background led to a job inspecting fuses at a munitions factory. Later he worked as a health inspector, checking water supplies and kitchens at Miami Beach hotels earmarked for pilots in the Army Air Corps.

When the war ended Prusoff traveled to New York to study at Columbia University, where he worked in the laboratory of Charles Glen King, Ph.D., studying the relationship of folic acid to nucleic acid biosynthesis.

At Columbia, Prusoff noticed a familiar name on a list of students. He had met Brigitte Auerbach, a chemist at Merck and Co., when he worked in Tennessee as an ordnance inspector. They eventually married and had two children, Laura, 45, now a photographer living in Turkey, and Alvin, 47, a father of three, who is a computer consultant, active in civic affairs and a coach in Fairfield County sports leagues. Brigitte Prusoff launched her own distinguished career in epidemiology and biostatistics at Yale. After her death in 1991, Prusoff endowed a lecture series in her honor at Epidemiology/Public Health and Psychiatry.

Prusoff received his doctorate from Columbia in 1949 and went to Western Reserve University to work in the laboratory of Arnold D. Welch, M.D., Ph.D. Welch, a rising star in pharmacology, would later reinvigorate the pharmacology department at Yale. It was with Welch that Prusoff made both his first major contribution and first major blunder in pharmacology.

The two men were trying to purify the intrinsic factor. This natural protein is found in gastric juices. It is necessary for the absorption of vitamin B12 from the gastrointestinal tract, which, in turn, helps the body stave off pernicious anemia. “We reasoned that if we were able to isolate and characterize the intrinsic factor, this could be given to patients who have pernicious anemia,” he says. A Harvard colleague, William Castle, M.D., whom Prusoff calls the father of the intrinsic factor, sent Welch two liters of gastric juices pumped from the stomachs of medical students. Prusoff put the fluid through a vacuum freeze-drying process that separated the liquid, leaving a powder that contained the intrinsic factor. His next task was to release the vacuum.

“When I released the vacuum I did it too fast and all the powder went into the condenser, with no way to recover it,” Prusoff says. “I had to go in and tell Dr. Welch what I had done. I said if he threw me out of the laboratory he would have been justified. He felt my remorse, and I guess had confidence in me and didn’t fire me.” Despite the mishap, Prusoff succeeded in obtaining a 5,000-fold purification and characterizing the intrinsic factor. Laughing, he adds, “That’s when we changed direction and went into nucleic acid chemistry.”

With Welch, he came to Yale in 1953. Here, Prusoff began what has become a lifelong quest — ways to modify thymidine’s structure to curb disease. Even now, Prusoff is collaborating with researchers in Moscow and at Washington State University on two anti-HIV projects, one of them a thymidine analog. The other is based upon his hunch that the boronated peptides will inhibit HIV protease and prevent the formation of infectious viral particles. He and his colleague, Arman Pivazyan, Ph.D., an associate research scientist in pharmacology, are working on their second generation of the peptides. Unlike other protease inhibitors on the market, the boronated peptides not only attack the protease enzyme at its active site, but also prevent two enzyme subunits from binding and becoming active. “We hope, because the mechanism of action of these compounds is different, HIV will be less resistant to them,” says Pivazyan, who came to Yale from Moscow in 1990 to work with Prusoff. “We have shown that these compounds act as dual-mode inhibitors. We have shown that mutated protease is less resistant to this compound than commercial protease inhibitors.” Now he and Prusoff are working on increasing the potency of the compound.

Pivazyan describes their working relationship as informal. He and Prusoff, he says, are two scientists working together. Pivazyan works alone in the laboratory and consults frequently with Prusoff, who comes to his office every day. “I know what the general problem is,” Pivazyan says. “I work on the problem and we discuss the results and what to do next.”

Prusoff and Pivazyan have applied for an NIH grant to continue their work. They expect it will take several years and many more generations of their compound before it is ready for in vivo tests. Once they determine its potency against HIV protease, it will go to the laboratory of Prusoff’s old friend and colleague Cheng, who will conduct further tests of its effect on HIV. Cheng and Prusoff first met in the early 1970s, when Cheng arrived from Brown University to begin his postdoctoral work. He chose to study at Yale because of Prusoff. “He was a leading authority in the antiviral area. He was a leading authority in the area of pyrimidine nucleoside analogs,” Cheng recalled. “Most importantly, he has such a nice personality. I saw him as a role model.”

Cheng went on to develop 3TC for use against AIDS and is responsible for five of the 10 Yale-developed compounds now in pre-clinical or clinical trials. He considers Prusoff a giant in his field. “In general, you make one major break and add other things to it,” says Cheng, who goes on to cite Prusoff’s development of idoxuridine, the idoxuridine amino analog and d4T. “He has made three major breaks.”

Recognizing his lifetime of accomplishment, the medical school has created the William H. Prusoff Chair in Pharmacology. Dean David A. Kessler, M.D., announced the chair at a symposium in Prusoff’s honor last year. After the symposium, colleagues and friends feted him with a dinner and accolades that touched on his character as well as his achievements. As one attendee remarked, the crowd included “anyone who is anyone in pharmacology.”

“I am very, very lucky that my whole career has been a very happy one,” Prusoff says. Along with his family, his work has been the driving force in his life. He abandoned his only hobby, traveling, eight years ago when Brigitte died. Although retired, he spends time every day in his office. “It’s better than staying home and contemplating one’s navel,” he says. “The important thing is that I enjoy the work. It has been a very happy and productive career.”