The Resistance Part I:
From Petri Dishes to Population Dynamics

By Jason Socrates Bardi

"The air was free from gnats, the earth from weeds or fungi; everywhere were fruits and sweet and delightful flowers; brilliant butterflies flew hither and thither. The ideal of preventive medicine was attained. Diseases had been stamped out."

—H.G. Wells, The Time Machine, 1898.

When the history of science and medicine in the 20th century is eventually written, human immunodeficiency virus (HIV) will likely get an entire solemn chapter. Emerging in the early 1980s, ironically just after the major public health victory against smallpox, HIV had spread to all corners of the globe by the end of the century.

Today, the disease shows no signs of abating. There is no vaccine to prevent the spread of HIV and no cure for AIDS, the disease the virus engenders.

Science and medicine, however, have had their share of successes controlling HIV. The rise of antiretroviral drugs in 1980s and 1990s proved that AIDS could be a treatable disease. These drugs, which target virus-specific enzymes like HIV protease and reverse transcriptase, showed great efficacy at keeping the virus in check, which often dramatically improved the prognosis of those infected. They also provided a relatively inexpensive way of reducing mother-to-child transmission of the virus. These are significant breakthroughs, and there are many people alive today who owe their lives to antiretrovirals.

The virus has fought back, however, with the emergence of strains resistant to these drugs.

The Coming Resistance

Now a large research consortium that brings together investigators, students, postdocs, and other scientists at The Scripps Research Institute (TSRI) with their colleagues at several other institutions is beginning to use resistant HIV protease to develop methodologies for drug evolution.

Led by Molecular Biology Professor Arthur Olson, the group seeks to establish a drug design "cycle" aimed at developing, testing, and refining novel approaches to making specific inhibitors of HIV protease that would be capable of limiting or eliminating drug resistance.

The group is trying to understand resistance by looking at what happens as the virus changes in response to protease inhibitors. They are looking to identify the sequential protease transition mutants and to understand their most basic to advanced biology, including sequence, biochemical reactivity, and structure. The researchers are also looking at resistance as a phenomenon—how it can be predicted to how it can be countered.

"How do these changes work at the atomic level?" asks Associate Professor Bruce Torbett, one of the investigators in this consortium, which is funded by a program project grant from the National Instuitutes of Health called Drug Design Cycle Targeting HIV-Protease Drug Resistance.

Rather than aiming solely to make the next useful AIDS drug—which is naturally one goal of all the investigators—the team is seeking to bring all of its knowledge and technology to bear on developing a methodology that will allow them to understand something much more complex.

"We want to be able to predict what really happens when [a patient] takes a drug," says Torbett. "We're trying to figure out how mutable the protease is biologically, structurally, chemically, and we're asking what are the possible mutations, when those mutations happen, and what they do in terms of fitness of the virus."

In particular, the researchers are focusing on active site mutations. There are 10 amino acids in the binding site of the protease that have contact with the protein chain that the protease cleaves (in actuality 20, since the protease is a dimer composed of two identical 99 amino acid chains).

"The way most people design drugs is to fill up the [binding] site and get the most binding energy," says Olson. "But certain mutations can easily knock [these drugs] out."

For instance, a mutation that substitutes a phenylalanine for almost any other amino acid among the 10 in the active site of the HIV protease enzyme will add a significant amount of bulk. This added bulk will effectively hinder any drug that was designed to fill the binding site.

The goal for the next several years, says Olson, is to ask questions to get a sense of the protease's ability to mutate. What are the rules the virus follows in acquiring mutations?

Can one develop a model that will predict how the virus will respond to various drug regimens? Are there compounds that could effectively box the protease in?

The Cat's in that Corner

The program project grant has a long history at TSRI and is currently beginning its third round of funding. The first round, in the early 1990s, was primarily concerned with comparing HIV to its cousin, the feline immunodeficiency virus (FIV).

FIV was discovered in California in 1986 by Niels Pedersen, who is currently director of the Center for Companion Animal Health at the University of California, Davis, and Janet Yamamoto, who is now a professor in the University of Florida's College of Veterinary Medicine.

As the story goes, there was a kindly woman who took in strays—many strays—housing them in her large kennels. She noticed an odd thing. Several cats under her care became sick and eventually died, seemingly as a result of sleeping in the same pen as one particular feral cat. So she contacted Pedersen, who took samples and eventually isolated a virion, which under the electron microscope looked like an RNA virus belonging to the lentivirus family.

Shortly thereafter, TSRI Professor John Elder, who had been working on retroviruses for 10 years by 1986, began to collaborate with the Pedersen laboratory to work on the virus taken from that isolate and from another isolate from a cat belonging to former TSRI investigators Fred Hefron and Maggie So.

"They had a cat that came down with FIV, and we isolated the virus from it," says Elder.

When they started, not much was known about the structure of the FIV protease. In the first round of funding, Elder and his colleagues isolated, cloned, and purified proteases for their crystallographer collaborators to solve. Their hope was that their discoveries about FIV would shed light on the problem of HIV.

The highlight of the second round of funding was the development of the TL3 inhibitor, which could effectively inhibit both HIV and FIV.

FIV and HIV, it turned out, are closely related, which makes FIV a good model for studying an HIV-type infection. HIV and FIV proteases have a 32 percent amino acid identity and essentially the same three-dimensional structure.

"If I showed you two pictures of FIV protease and HIV protease, you couldn't tell them apart," says Elder.

Yet, strangely, the two proteases respond differently to the same inhibitors. Common drugs that inhibit HIV protease do not work on the FIV protease at all.

This observation led Elder, in collaboration with TSRI investigators Chi-Huey Wong, who is the Ernest W. Hahn Professor and Chair in Chemistry, Torbett, Olson, and several others, to ask what subtle differences between the structures of FIV protease and HIV protease could cause such a great distinction.

It was Research Associate Taekyu Lee of Wong's group who noticed a region of the FIV protease that was smaller than the corresponding region in the HIV protease.

"We looked at the sequences and structures of all the known mutants," says Wong. "We found that there was a common trend in the mutants [whereby] one site becomes smaller and smaller."

This reduction is in what is known as the P3 binding site of the protease—where the enzyme makes contact with one end of the inhibitor and where it normally would interact with residues of the protein chain it naturally cleaves three amino acids down from where the protease breaks the chain. Most commercial HIV protease inhibitors, says Wong, are designed to fill a large P3 site, but the virus often acquires resistance to these drugs when it mutates the amino acids in the P3 site swapping small amino acids for bulky ones. The team recognized this, and the knowledge helped them design a new inhibitor to fit this smaller space.

This prompted Wong to replace the inhibitor residue that fits in that site with a smaller amino acid. When they did this, the potency of this inhibitor increased 1,000-fold for FIV.

"This was the best [inhibitor] we had ever seen against FIV," says Elder, adding that it was also efficacious against the wild-type HIV and nine out of thirteen protease-resistant HIV isolates tested. The molecular changes that allowed many variants of HIV to escape drug therapies were the same as those that made FIV distinct from HIV.

"That observation told us that we could use FIV protease as a drug resistant model for HIV protease," says Wong.

The current project spans various aspects of the design, synthesis, and analysis of compounds to target the HIV protease, mutant proteases, and possible related targets, like RNA. In one sense, the project is focused on drug design and has all the classic components of such a project—including structural biologists and modelers, molecular biologists with expertise in gene expression; synthetic organic chemists who can make new lead compounds; and biologists who can test them in cell culture and other model systems. However, the project is not so straightforward.

"We are actually funded to develop methodology—not to cure AIDS," says Olson, adding that AIDS is only one disease out of many in which the evolution of resistance to drugs threatens to make these diseases harder and more expensive to treat.

The program project grant is organized around four collaborative projects supported by two cores. In general terms, the groups involved in the projects analyze the effect of HIV mutations on various protease inhibitors computationally and experimentally in a coordinated and interactive effort.

Olson, along with TSRI Associate Professor David Goodsell and University of California, San Diego's Department of Cognitive Science Professor Richard Belew, is taking a computational "coevolutionary" approach to develop models of mutant forms of the HIV protease and predict the interactions of these inhibitors against all the mutant protein.

Torbett's project involves using biological models like tissue culture. These assays help to derive the computer models, which can then be used to predict the results of further biological assays, and these assays can then be tested and used to improve the computer model in an iteratively improving process. Torbett's project will also interact closely with the two other projects in the grant, which are led by Wong and 2001 Nobel Laureate K. Barry Sharpless, who is the W.M. Keck Professor of Chemistry at TSRI.

Sharpless, joined by Associate Professor M.G. Finn and Assistant Professor Valery Fokin are applying their in situ click chemistry for the rapid development and evolution of inhibitors to drug-resistant proteases.

Wong is taking a synthetic chemical approach to designing inhibitors of HIV protease. He is also interested in using his methodology to target RNA structures associated with the protease, believing that there may be some targets there that are not as susceptible to mutation.

All four projects interact with each other and alternatively suggest experiments and take suggestions from one another. And they also interact closely with two different scientific cores funded by the grant.

Protein Expression and Crystallization Cores

One of the two cores of the program project grant is led by Elder and Assistant Professor Philip Dawson. This Protein Expression Core, through the efforts of Research Associate Ying-Chuan Lin of the Elder lab, produces proteases and mutant forms of the protease, using a combination of synthetic chemistry and biological expression systems. They also design functional assays and chemical probes to see how mutations affect the activity of the enzyme. And they look at the substrate specificity of the HIV protease, working with substrate and substrate-like inhibitors.

"In general," says Dawson, "our role is to apply the technologies that we have been developing to the study of drug resistance."

Dawson and his colleagues use the technique of solid-phase synthesis to make the peptides. Invented by Robert Bruce Merrifield in 1963 (for which he was awarded the 1984 Nobel Prize in Chemistry), solid phase protein synthesis basically entails building a peptide step-by-step, starting with a single amino acid that is attached to a polymer resin. Amino acids are then added one at a time, the resin is washed between each successive round, and finally the finished peptide is removed from the resin.

This expertise in chemically synthesizing proteins also gives Dawson and his laboratory the ability to routinely make proteins for a number of applied problems. One problem they work on is the protease encoded by HIV.

Dawson joined the program project grant during the latest round of funding. His expertise in chemically synthesizing proteins gives them the ability to routinely make proteins for a number of applied problems. Actually, says Dawson, "HIV protease is one of the big success stories of chemical protein synthesis." The enzyme was synthesized and then crystallized thanks to the work of Dawson's Ph.D. advisor and former TSRI Professor Steve Kent (now at the University of Chicago) and Alexander Wlodawer, who is currently chief of the Macromolecular Crystallography Laboratory and the Protein Structure Section at the National Cancer Institute.

Back in the late 1980s, when the push to design potent inhibitors of HIV protease was in full swing, one major obstacle was expressing and purifying the protein. Kent successfully synthesized HIV protease and Wlodawer successfully crystallized it and solved its three-dimensional structure. This work led to the structure-assisted design of a number of drugs.

Wlodawer is also involved as one of the co-lead investigators on the second of the program project grant's two cores. This core is devoted to elucidating the structure and modeling of various forms of HIV protease. These structures also provide important information to Olson, who can use them as the starting point for designing lead compounds, which can then be handed off to the chemists, who can use them as guides for synthesizing new inhibitors. And any compounds that they do develop can then be tested by Torbett.

TSRI Associate Professor Dave Stout is head of this core, and he is leading an effort to crystallize the proteins with RNA in order to look at RNA–protease interactions. Additionally, this core runs computational protein–inhibitor docking experiments and other computer aided drug design studies.

It's a very dynamic group of people, says Fokin. "It's always interesting to see what other people come up with."

Populating Dynamics and Dimer Inhibitors

There are many interesting questions being raised within the program project grant, including some that take quite unusual approached to addressing HIV protease resistance.

For instance, Goodsell ties atomic models to population models.

"You can create a model that estimates all the cells in the immune system, how they reproduce, and how the virus interacts with them," says Goodsell. This allows him to ask such questions as: If a patient takes one particular AIDS drug for a year, what would the population of mutants in his/her body be like at the end of that year? Would being able to predict this generate better-tailored therapies or point the way to the best possible combination of therapies?

Another unusual approach is asking if parts of the protease molecule other than the active site can be targeted with drugs. Specifically, can one target what is known as the "dimer interface" of the molecule, which is where the two ends of two HIV protease monomers come together and form a beta-sheet—like interlaced fingers from two hands that hold the active dimer form of the molecule together.

The aim is to target this interface and prevent two individual protease monomers from coming together. Since the protease enzyme is only active as a dimer, mucking with this dimer interface should profoundly affect the activity of the enzyme.

"If we target those areas, what does the protease do?" asks Torbett.

Of course, the problem of mutations to the protease cannot be avoided here either. Not all of the mutations that arise in the protease enzyme are in the active site or are functionally related to the active site. In fact, many of the mutants that have been raised by the TSRI team in the laboratory and that have arisen in patients who are taking antiretrovirals are not in the active site of the protease. Some are in the area of this dimer interface and altering the stability of the dimer.

One interesting way that they are looking at this is by forcing dimerization of the protease monomers by expressing them together in a linked form.

Next Week: Fighting HIV Resistance at Home and in the Laboratory

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