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The Structures of Life

Chapter 4: Structure-Based Drug Design: From the Computer to the Clinic

In 1981, doctors recognized a strange new disease in the United States. The first handful of patients suffered from unusual cancers and pneumonias. As the disease spread, scientists discovered its cause—a virus that attacks human immune cells. Now a major killer worldwide, the disease is best known by its acronym, AIDS.

AIDS or acquired immunodeficiency syndrome, is caused by the human immunodeficiency virus, or HIV.

Although researchers have not found a cure for AIDS, structural biology has greatly enhanced their understanding of HIV and has played a key role in the development of drugs to treat this deadly disease.

The Life of an AIDS Virus

Illustration courtesy of Louis E. Henderson, Senior Scientist (emeritus, retired) AIDS Vaccine Program, National Cancer Institute (Frederick, MD)
Illustration courtesy of Louis E. Henderson, Senior Scientist (emeritus, retired) AIDS Vaccine Program, National Cancer Institute (Frederick, MD)
Click for larger image

HIV was quickly recognized as a retrovirus, a type of virus that carries its genetic material not as DNA, as do most other organisms on the planet, but as RNA. After entering a cell, retroviruses "reverse transcribe" their RNA into DNA.

Long before anyone had heard of HIV, researchers in labs all over the world studied retroviruses, some of which cause cancers in animals. These scientists traced out the life cycle of retroviruses and identified the key proteins the viruses use to infect cells.

When HIV was identified as a retrovirus, these studies gave AIDS researchers an immediate jump-start. The previously identified viral proteins became initial drug targets.

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Revealing the Target

HIV protease is a symmetrical molecule with two equal halves and an active site near its center. Molecular models of HIV protease in this chapter were generated by Alisa Zapp Machalek
HIV protease is a symmetrical molecule with two equal halves and an active site near its center. Molecular models of HIV protease in this chapter were generated by Alisa Zapp Machalek

Our story begins in 1989, when scientists determined the X-ray crystallographic structure of HIV protease, a viral enzyme critical in HIV's life cycle. Pharmaceutical scientists hoped that by blocking this enzyme, they could prevent the virus from spreading in the body.

With the structure of HIV protease at their fingertips, researchers were no longer working blindly. They could finally see their target enzyme—in exhilarating, color-coded detail. By feeding the structural information into a computer modeling program, they could spin a model of the enzyme around, zoom in on specific atoms, analyze its chemical properties, and even strip away or alter parts of it.

Knowing that HIV protease has two symmetrical halves, pharmaceutical researchers initially attempted to block the enzyme with symmetrical small molecules. They made these by chopping in half molecules of the natural substrate, then making a new molecule by fusing together two identical halves of the natural substrate.
Knowing that HIV protease has two symmetrical halves, pharmaceutical researchers initially attempted to block the enzyme with symmetrical small molecules. They made these by chopping in half molecules of the natural substrate, then making a new molecule by fusing together two identical halves of the natural substrate.

Most importantly, they could use the computerized structure as a reference to determine the types of molecules that might block the enzyme. These molecules can be retrieved from chemical libraries or can be designed on a computer screen and then synthesized in a laboratory. Such structure-based drug design strategies have the potential to shave off years and millions of dollars from the traditional trial-and-error drug development process.

These strategies worked in the case of HIV protease inhibitors. “I think it's a remarkable success story,” says Dale Kempf, a chemist involved in the HIV protease inhibitor program at Abbott Laboratories. “From the identification of HIV protease as a drug target in 1988 to early 1996, it took less than 8 years to have three drugs on the market.” Typically, it takes 10 to 15 years and more than $800 million to develop a drug from scratch.

Illustration of butterfliesThe structure of HIV protease revealed a crucial fact—like a butterfly, the enzyme is made up of two equal halves. For most such symmetrical molecules, both halves have a "business area," or active site, that carries out the enzyme's job. But HIV protease has only one such active site—in the center of the molecule where the two halves meet.

Pharmaceutical scientists knew they could take advantage of this feature. If they could plug this single active site with a small molecule, they could shut down the whole enzyme—and theoretically stop the virus' spread in the body.

Several pharmaceutical companies started out by using the enzyme's shape as a guide. “We designed drug candidate molecules that had the same two-fold symmetry as HIV protease,” says Kempf. “Conceptually, we took some of the enzyme's natural substrate [the molecules it acts upon], chopped these molecules in half, rotated them 180 degrees, and glued two identical halves together.”

To the researchers' delight, the first such molecule they synthesized fit perfectly into the active site of the enzyme. It was also an excellent inhibitor—it prevented HIV protease from functioning normally. But it wasn't water-soluble, meaning it couldn't be absorbed by the body and would never be effective as a drug.

Illustration of the various components of a good medicine: the best possible activity, solubility, bioavailability, half-life, and metabolic profile
A drug candidate molecule must pass many hurdles to earn the description "good medicine." It must have the best possible activity, solubility, bioavailability, half-life, and metabolic profile. Attempting to improve one of these factors often affects other factors. For example, if you structurally alter a lead compound to improve its activity, you may also decrease its solubility or shorten its half-life. The final result must always be the best possible compromise.

Abbott scientists continued to tweak the structure of the molecule to improve its properties. They eventually ended up with a nonsymmetrical molecule they called Norvir® (ritonavir).

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A Hope for the Future

Between December 1995 and March 1996, the Food and Drug Administration approved the first three HIV protease inhibitors—Hoffman-La Roche's Invirase™ (saquinavir), Abbott's Norvir™ (ritonavir), and Merck and Co., Inc.'s Crixivan® (indinavir). Initially, these drugs were hailed as the first real hope in 15 years for people with AIDS. Newspaper headlines predicted that AIDS might even be cured.

Although HIV protease inhibitors did not become the miracle cure many had hoped for, they represent a triumph for antiviral therapy. Antibiotics that treat bacterial diseases abound (although they are becoming less effective as bacteria develop resistance), but doctors have very few drugs to treat viral infections.

Protease inhibitors are also noteworthy because they are a classic example of how structural biology can enhance traditional drug development. “They show that with some ideas about structure and rational drug design, combined with traditional medicinal chemistry, you can come up with potent drugs that function the way they're predicted to,” says Kempf.

“That doesn't mean we have all the problems solved yet,” he continues. “But clearly these compounds have made a profound impact on society.” The death rate from AIDS went down dramatically after these drugs became available. Now protease inhibitors are often prescribed with other anti-HIV drugs to create a "combination cocktail" that is more effective at squelching the virus than are any of the drugs individually.

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Structure-Based Drug Design: Blocking the Lock

Traditional drug design often requires random testing of thousands—if not hundreds of thousands—of compounds (shown here as metal scraps)
Traditional drug design often requires random testing of thousands—if not hundreds of thousands—of compounds (shown here as metal scraps)

Traditionally, scientists identify new drugs either by fiddling with existing drugs or by testing thousands of compounds in a laboratory. If you think of the target molecule—HIV protease in this case—as a lock, this approach is rather like trying to design a key perfectly shaped to the lock if you're given an armload of tiny metal scraps, glue, and wire cutters.

Using a structure-based strategy, researchers have an initial advantage. They start with a computerized model of the detailed, three-dimensional structure of the lock and of its key (the natural molecule, called a substrate, that fits into the lock, triggering viral replication). Then scientists try to design a molecule that will plug up the lock to keep out the substrate key.

Knowing the exact three-dimensional shape of the lock, scientists can discard any of the metal scraps (small molecules) that are not the right size or shape to fit the lock. They might even be able to design a small molecule to fit the lock precisely. Such a molecule may be a starting point for pharmaceutical researchers who are designing a drug to treat HIV infection.

By knowing the shape and chemical properties of the target molecule, scientists using structure-based drug design strategies can approach the job more 'rationally.' They can discard the drug candidate molecules that have the wrong shape or properties.
By knowing the shape and chemical properties of the target molecule, scientists using structure-based drug design strategies can approach the job more "rationally." They can discard the drug candidate molecules that have the wrong shape or properties.

Of course, biological molecules are much more complex than locks and keys, and human bodies can react in unpredictable ways to drug molecules, so the road from the computer screen to pharmacy shelves remains long and bumpy.

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Homing in on Resistance

Scientists have identified dozens of mutations (shown in red) that allow HIV protease to escape the effects of drugs. The protease molecules in some drug-resistant HIV strains have two or three such mutations. To outwit the enzyme's mastery of mutation, researchers are designing drugs that interact specifically with amino acids in the enzyme that are critical for the enzyme's function. This approach cuts off the enzyme's escape routes. As a result, the enzyme-and thus the entire virus-is forced to succumb to the drug.
Scientists have identified dozens of mutations (shown in red) that allow HIV protease to escape the effects of drugs. The protease molecules in some drug-resistant HIV strains have two or three such mutations. To outwit the enzyme's mastery of mutation, researchers are designing drugs that interact specifically with amino acids in the enzyme that are critical for the enzyme's function. This approach cuts off the enzyme's escape routes. As a result, the enzyme—and thus the entire virus—is forced to succumb to the drug.

HIV is a moving target.When it reproduces inside the body, instead of generating exact replicas of itself, it churns out a variety of slightly altered daughter virus particles. Some of these mutants are able to evade, or "resist," the effects of a drug—and can pass that resistance on to their own daughter particles.While most virus particles initially succumb to the drug, these resistant mutants survive and multiply. Eventually, the drug loses its anti-HIV activity, because most of the virus particles in the infected person are resistant to it.

Some researchers now are working on new generations of HIV protease inhibitors that are designed to combat specific drug-resistant viral strains.

Detailed, computer-modeled pictures of HIV protease from these strains reveal how even amino acid substitutions far away from the enzyme's active site can produce drug resistance. Some research groups are trying to beat the enzyme at its own game by designing drugs that bind to these mutant forms of HIV protease. Others are designing molecules that latch onto the enzyme's Achilles' heels—the aspartic acids in the active site and other amino acids that, if altered, would render the enzyme useless. Still others are trying to discover inhibitors that are more potent, more convenient to take, have fewer side effects, or are better able to combat mutant strains of the virus.

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How HIV Resistance Arises

HIV produces many different versions of itself in a patient's body (although the huge majority are the normal form).
HIV produces many different versions of itself in a patient's body (although the huge majority are the normal form).
Drugs kill all of these virus particles except those that are resistant to the drugs.
Drugs kill all of these virus particles except those that are resistant to the drugs.
The resistant virus particles continue to reproduce. Soon the drug is no longer effective for the patient.
The resistant virus particles continue to reproduce. Soon the drug is no longer effective for the patient.

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Gripping Arthritis Pain

While the HIV protease inhibitors are classic examples of structure-based drug design, they are also somewhat unusual—at least for now. Although many pharmaceutical companies have entire divisions devoted to structural biology, most use it as a complementary approach, in partnership with other, more traditional, means of drug discovery. In many cases, the structure of a target molecule is determined after traditional screening, or even after a drug is on the market.

Rheumatoid arthritis is an immune system disorder that affects more than 2 million Americans, causing pain, stiffness, and swelling in the joints. It can cripple hands, wrists, feet, knees, ankles, shoulders, and elbows. It also causes inflammation in internal organs and can lead to permanent disability. Osteoarthritis has some of the same symptoms, but it develops more slowly and only affects certain joints. National Institutes of Health
Rheumatoid arthritis is an immune system disorder that affects more than 2 million Americans, causing pain, stiffness, and swelling in the joints. It can cripple hands, wrists, feet, knees, ankles, shoulders, and elbows. It also causes inflammation in internal organs and can lead to permanent disability. Osteoarthritis has some of the same symptoms, but it develops more slowly and only affects certain joints. National Institutes of Health

This was the case for Celebrex®. Initially designed to treat osteoarthritis and adult rheumatoid arthritis, Celebrex® became the first drug approved to treat a rare condition called FAP, or familial adenomatous polyposis, that leads to colon cancer.

Normally, the pain and swelling of arthritis are treated with drugs like aspirin or Advil® (ibuprofen), the so-called NSAIDs, or non-steroidal anti-inflammatory drugs. But these medications can cause damage to gastrointestinal organs, including bleeding ulcers. In fact, a recent study found that such side effects result in more than 100,000 hospitalizations and 16,500 deaths every year. According to another study, if these side effects were included in tables listing mortality data, they would rank as the 15th most common cause of death in the United States.

A fortunate discovery enabled scientists to design drugs that retain the anti-inflammatory properties of NSAIDs without the ulcer-causing side effects.

By studying the drugs at the molecular level, researchers learned that NSAIDs block the action of two closely related enzymes called cyclooxygenases. These enzymes are abbreviated COX-1 and COX-2.

 

Illustration of prostaglandins in human body.
Both COX-1 and COX-2 produce prostaglandins, which have a variety of different—and sometimes opposite—roles in the body. Some of these roles are shown here.

Although the enzymes share some of the same functions, they also differ in important ways. COX-2 is produced in response to injury or infection and activates molecules that trigger inflammation and an immune response. By blocking COX-2, NSAIDs reduce inflammation and pain caused by arthritis, headaches, and sprains.

In contrast, COX-1 produces molecules, called prostaglandins, that protect the lining of the stomach from digestive acids.When NSAIDs block this function, they foster ulcers.

To create an effective painkiller that doesn't cause ulcers, scientists realized they needed to develop new medicines that shut down COX-2 but not COX-1. Such a compound was discovered using standard medicinal chemistry and marketed under the name Celebrex®. It quickly became the fastest selling drug in U.S. history, generating more prescriptions in its first year than the next two leading drugs combined.

At the same time, scientists were working out the molecular structure of the COX enzymes. Through structural biology, they could see exactly why Celebrex® plugs up COX-2 but not COX-1.

The three-dimensional structures of COX-2 and COX-1 are almost identical. But there is one amino acid change in the active site of COX-2 that creates an extra binding pocket. It is this extra pocket into which Celebrex® binds.

In addition to showing researchers in atom-by-atom detail how the drug binds to its target, the structures of the COX enzymes will continue to provide basic researchers with insight into how these molecules work in the body.

Illustration of COC-1 and COX-2
Diagrams of the chemical makeup of Valine and Isoteucine.
This close-up view of the active sites of COX-1 and COX-2 (ribbons) reveal why Celebrex® can bind to one of the COX enzymes, but not to the other. A single amino acid substitution makes all the difference. In a critical place in the protein, COX-2 contains valine, a small amino acid that creates a pocket into which the drug (in yellow) can bind. In the same position, COX-1 contains isoleucine, which elbows out the drug. Adapted with permission from Nature ©1996 Macmillan Magazines Ltd.

 

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Student Snapshot: The Fascination of Infection

“I really like to study retroviruses,” says Kristi Pullen, who majored in biochemistry at the University of Maryland, Baltimore County (UMBC). “I also like highly infectious agents, like Ebola. The more virulent something is, the less it's worked on, so it opens up all sorts of fascinating questions. I couldn't help but be interested.”

Kristi Pullen
Courtesy of Kelly Burns Photography, Columbia, Maryland

In addition to her UMBC classwork, Pullen helped determine the structure of retroviruses in the NMR spectroscopy laboratory of Michael Summers. This research focuses on how retroviruses package "RNA warheads" that enable them to spread in the body. Eventually, the work may reveal a new drug target for retroviral diseases, including AIDS.

“Working in Dr. Summers' lab and other labs teaches you that research can be fun. It's not just a whole lot of people in white coats. We went biking and skiing together. All the people were great to work with.”
Kristi Pullen
Graduate Student
University of California, Berkeley

Until her senior year in high school, Pullen wanted to be an orthopedic surgeon. But after her first experience working in a lab, she recognized "there's more to science than medicine." Then, after taking some science courses, she realized she had an inner yearning to learn science and to work in a lab.

Pullen is now a graduate student at the University of California, Berkeley in the Department of Molecular and Cell Biology. She plans to continue studying structural biology, to earn a Ph.D., and possibly also to earn an M.D. She also has some longer-term goals.

“Ultimately what I want to do way, way, way down the line is head the NIH [National Institutes of Health] or CDC [Centers for Disease Control and Prevention] and in that way affect the health of a large number of people—the whole country.”

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Got It?

What is structure-based drug design?

How was structure-based drug design used to develop an HIV protease inhibitor?

How is the structural difference between COX-1 and COX-2 responsible for the effectiveness of Celebrex®?

How do viruses become resistant to drugs?

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This page last reviewed on October 27, 2011