The Resistance Part III:
Chemical Innovations and New Approaches

By Jason Socrates Bardi

"Perhaps the eye of a scrutinizing observer might have discovered a barely perceptible fissure, which, extending from the roof of the building in front, made its way down the wall in a zigzag direction"

—Edgar Allen Poe, The Fall of the House of Usher, 1839.

Human immunodeficiency virus (HIV) plays a simple game of overwhelming numbers.

The game is analogous to what would take place in an honest casino that has been infiltrated by thousands of crooked gamblers who are trying to break the bank. The gamblers constantly try different strategies of cheating, until eventually the house loses.

Imagine that the casino tries to control these losses by hiring private detectives who walk around the casino floor and grab any crooked gamblers they see, incapacitating them and preventing them from cheating. But the crooked gamblers soon learn how to outwit the house detectives by disguising their faces so that they are no longer recognized.

The same thing happens with HIV.

The virus replicates prolifically in the body, and has such a high frequency of mutation that it can rapidly evolve resistance to the drugs that are used to treat it. A scientific consortium led by Professor Arthur Olson of The Scripps Research Institute (TSRI) was created to counter this HIV drug resistance.

Funded by a program project grant, the consortium combines the efforts of dozens of investigators, postdoctoral fellows, graduate students, and other researchers at TSRI and several other institutions. It seeks to establish a drug design cycle aimed at developing, testing, and refining novel approaches to making specific inhibitors that will disable resistant mutants of HIV protease, and it combines molecular and cellular biology, computational and structural approaches, and chemistry.

Two of the projects focus on the design and synthesis of compounds that inhibit HIV protease and drug-resistant strains of HIV protease. One of these two projects is led by TSRI Chemistry Professor Chi-Huey Wong.

A New Strategy

Against the wall in Wong's office is a shelf lined with volumes of chemistry and biology texts. Outside and down the hall is a reading room that has stacks of some of the leading journals in his field. Also in this reading room are several networked computers.

And yet when Wong and his colleagues began thinking of ways to combat HIV drug resistance several years ago, the answers were not to be found in any of these sources.

"We needed to have a new strategy," recalls Wong.

Wong is doing synthetic chemistry work for the project and coming up with new ways of targeting the virus, including using technology of his own design to identify unique spots in the viral mRNA that are highly conserved, even in the mutant strains. He has found that one good target is the RNA frameshift region of the virus.

Frameshifting, in biology, is something that takes place during the translation of the nucleotides into protein amino acids. It happens when a ribosome that is translating the RNA into protein shifts slightly, moving up or down the RNA sequence by a nucleotide or so. But this seemingly slight change can have a radical change on the amino acid sequence of the protein that is being translated—it may result in a completely different protein.

For instance, a sequence that originally is read: (UGG) (GCA) (UGU) (UGA) (CGU)... might be frameshifted into a sequence that would now be read: (...U) (GGG) (CAU) (GUU) (GAC)...

In HIV, the frameshift region is where the gag and pol regions of the viral RNA meet, and is highly conserved because frameshifting is necessary for the expression of the POL polyprotein, which contains the all-important viral enzymes, the reverse transcriptase, the integrase, and the protease. The virus cannot tolerate mutations to this region of RNA because mutations would block the frameshift and knock out the all-important viral enzymes.

Wong reasoned when he began this work that by designing an inhibitor that targets this non-mutating RNA, he could potentially have a compound that would be broadly effective against a wide variety of mutant HIV strains. In order to achieve this, he turned to the aminoglycosides.

One-Pot Synthesis

Aminoglycosides are compounds that interact with interact with RNA, and there are already drugs of this class on the market—like the antibiotic streptomycin, which targets specific pieces of RNA.

These drugs all target the RNA of one portion of the bacterial ribosome, known as the "30S subunit." The aminoglycosides bind to this RNA and prevent the ribosome from accurately translating protein. Wong reasoned that he might be able to use these compounds as scaffolds upon which to design a chemical that would specifically target the HIV mRNA frameshift region.

The basic technique that Wong uses is one he designed a few years ago for the synthesis of oligosaccharides in general, called programmable one-pot synthesis. This technique basically involves placing a large number of specific chemical building blocks into a reaction vessel and then making sequential chemical reactions in the soup. The final products depend entirely on the particular reaction scheme followed.

The one-pot technique, as Wong calls it, allows him to quickly assemble many types of carbohydrate structures—in just minutes or hours, depending on how complicated the chemistry is. Wong even developed software that he uses to streamline the process. "The computer will tell you which building blocks to use and you simply add them in sequence," he explains.

Depending on the type of building blocks used and the size of the final structures, the pool of compounds made in this way can be quite large. For instance, placing 300 building blocks in a pot and randomly combining them into tetramers will yield over eight billion compounds.

Once the oligosaccharides are made, these carbohydrate "libraries" can be used for any number of biological studies. One of Wong's main uses for the libraries is screening against various types of biological targets to look for interactions.

For instance, he created an array of several hundred aminoglycosides and screened for molecules that target HIV RNA by taking a 20- to 40-base piece of the viral RNA attached to a "biotin" molecule, which allows the bound aminoglycosides to be detected.

While this technology is promising, warns Wong, any potential drug that could be produced by this method still has to overcome the obstacle of cell permeability.

"If any [aminoglycoside compound] is going to be useful, it has to get into a cell," he says. While aminoglycosides are useful antibiotics already, they cannot get into human cells as easily as they can get into bacterial cells.

The eventual solution, says Wong, might be to develop a "prodrug" form of the compound, which would be converted to an active form after it enters a cell or to employ some "shuttle" molecules that can transport the drugs into the cells.

For now, he is at the beginning of this research. The structure of the program project grant is helpful because it allows him to use the structural and biochemical data that the other investigators on the project produce to help him come up with the molecular design of the molecules he is going to synthesize.

"Then you can go back to the biology and see if the chemistry works," says Wong. "An environment like Scripps is very good for a chemist. Through collaboration, you can solve a lot of interesting problems."

Click Chemistry

Together with TSRI Assistant Professor Valery Fokin, Professor K. Barry Sharpless, who is the W.M. Keck Professor of Chemistry, and Associate Professor M.G. Finn lead the second of the two chemically oriented projects on the grant.

"Our role is to develop and synthesize molecules that could potentially be inhibitors of HIV protease, and, using chemical tools, to learn more about the mutations of the protease," says Fokin. The technique they use is in situ click chemistry.

Click chemistry, a modular protocol for organic synthesis that Sharpless developed, is a powerful and original approach to drug design. In short, it relies on using energetic yet stable building blocks that will react with each other in a highly efficient and irreversible spring-loaded reaction. In its in situ variant, click chemistry uses the target enzyme itself to bring these building blocks together and to direct the formation of the desired inhibitor.

The idea is to use the HIV protease itself to design its own inhibitor by providing it with various building blocks. Only those building blocks that can form an inhibitor will be selected by the enzyme to "click" together. This technique has great potential to cut through a frenzy of possible inhibitors to demonstrate the best.

"We want to let the enzyme teach us what inhibitors [it prefers]," says Finn. "Those, in general, should be the better inhibitors."

The idea seems almost fantastic, but Sharpless and his colleagues have already had success with in situ click chemistry.

A few years ago, they published a paper in the scientific journal Angewandte Chemie describing the use of this technique to make a powerful inhibitor to acetylcholinesterase, a brain enzyme that breaks down acetylcholine, the neurotransmitter that propagates nerve signals. Inhibitors of acetylcholinesterase are used to treat the dementia associated with Alzheimer's disease, increasing the amount of acetylcholine in the brain, in turn enhancing brain activity. They have since expanded on this work using this method in several other systems.

As part of the program project grant, Sharpless and Finn want to see if they can apply the techniques of click chemistry to designing inhibitors of HIV protease.

"That enzyme," says Finn, "should, in principle, be amenable to the same kind of [click chemistry] strategy as acetylcholinesterase."

The strategy, Finn explains, is best applied to enzymes that have regions of protein-protein interfaces, such as proteins like HIV, where a dimer is formed by two identical protease monomers. Acetylcholinesterase itself is not a dimer, but it does have two binding regions adjacent to each other.

These protein-protein interfaces often have multiple potential binding sites for small molecules, and the trick with in situ click chemistry is to find classes of compounds that will bind tightly enough in the two faces of HIV that their proximity will allow their natural reactivity to take over.

Looking for a Short Cut

Employing the enzyme to make its own inhibitor could provide a great short cut. For instance, to take a simple case where inhibitors are made by combining two chemical structures—say one of 10 "A" structures and one of 10 "B" structures—then the possible number of structures multiplies.

With 10 possible "A" structures and 10 possible "B" structures, there would be 100 possible compounds. But with 100 possible "A" structures and 100 possible "B" structures, there would be 10,000 possible compounds.

"We don't have to make and screen all those," says Fokin. "We just have to allow the enzyme to select which ones fit best."

Inside the enzyme's binding pocket, the components should click together into a potent inhibitor of HIV protease, and once Sharpless, Fokin, and Finn recover the inhibitor, they can determine its structure and produce it in much larger quantities so that their colleagues on the program project grant can study the interaction of this inhibitor with the protease in structural and tissue culture experiments.

As the cycle continues, the way that the TSRI researchers envision it, other members of the project will provide mutant and wild type protease, and the Sharpless, Fokin and Finn laboratories will then repeat the process.

Dynamic Therapy

At the moment, this project is still in the early stages.

Together with the computational team, Sharpless, Finn, and Fokin are deciding on the best building blocks to start with. The Sharpless group has also been developing a benign copper catalyst and a methodology for copper-catalyzed "stitching" of azide and alkyne building blocks that will allow them to make a variety of the inhibitor analogs they are interested in. In addition to generating libraries of the analogs, the technique could also be used to produce large amounts of click inhibitors for further studies.

"Everything changed when we discovered the copper-catalyzed process for the synthesis of triazoles," says Fokin. "It makes the whole process go a lot smoother."

Another intriguing question they are asking is whether they can possibly apply the technique of in situ click chemistry in vivo.

In vivo means literally "in life," which in biology is generally understood to apply to experiments that take place in a living organism. In this case, in vivo in situ click chemistry suggests the development of a new type of cocktail—one that is made inside the target enzymes inside a living organism.

The idea is to give a cocktail of building blocks to a patient from which the final structure of the best inhibitor will be made. Only those building blocks that are effective against the protease enzymes encoded by the particular strain of HIV that infects that one patient would react and make inhibitors. Another intriguing alternative is to provide a pharmacist with a collection of building blocks, or "pre-drugs", which can be dispensed to each patient based on the specific information about the mutation.

"We asked, 'Can we actually apply click chemistry to treatment?'" says Fokin. "'Can we use our bodies to decide what kind of inhibitors to make?'"

The idea is that the therapy would be flexible enough so that it would work no matter which strains of HIV infect a person. By giving patients the subunits, the particular drug needed at that moment would be selected by whichever strain of HIV infected them.

While these ideas are tantalizing, they are a long way from becoming a reality. The investigators are still doing the first in situ studies with HIV, and any eventual in vivo studies would have to be done in cells first and then in model systems, before extensive human trials for safety and efficacy could even begin.

Still, says Fokin, the concept as they envisioned it offers a new way to approach what is now the long-standing problem of HIV drug resistance.



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