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.


See: HIV Pathogenesis and Drug Resistance

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.


Next Page | The Third Round of the Program Project Grant

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Assistant Professor Philip Dawson says, "In general, our role is to apply the technologies that we have been developing to the study of drug resistance." Photo by Jason S. Bardi.


Professor John Elder works with a cat virus, FIV, that is closely related to the human virus HIV. Photo by Jason S. Bardi.


Associate Professor David Goodsell is one of the investigators taking a computational approach to developing models of mutant HIV proteins. Self-portrait by David Goodsell.


Associate Professor Dave Stout is leading an effort to crystallize proteins with RNA in order to look at RNA-protease interactions. Photo by Kevin Fung.