Assembly inhibitors offer hope for future treatment

Researchers explain their work in this area 

Some very early research into a nontraditional target holds promise for a new line of defense against HIV in decades to come. New compounds that are tentatively being called maturation or assembly inhibitors provide a very early target in HIV’s activity within the body, researchers say.

Protease inhibitors block the cleavage and have been extremely successful therapeutically, says Peter Prevelige, PhD, professor of microbiology at the University of Alabama, Birmingham.

"But data suggest that you need both cleavage and rearrangement, and so another target is blocking the rearrangement," he says.

"In order to do that, you need to know what the action is between subunits in the rearranged, mature form, and that’s been difficult to do because viruses are not amenable to traditional, structural biology techniques, such as crystography and electron microscopy and imaging construction." Prevelige and co-investigators have developed a technique called mass spectrometry to identify those interfaces, Prevelige says.

"And we identified an interface formed from immature virus to mature virus, and that’s a potential drug target," he adds. "The category of compounds would be something like maturation inhibitors or assembly inhibitors."

To beat the virus, researchers need to continually identify new targets, Prevelige notes.

"The idea behind the new potential new class of assembly inhibitors is this: Proteins in the virus are translated into a long string of proteins linked together and HIV’s enzyme called the protease cuts this precursor protein into an individual mature protein, which is then liberated," says Andrew H. Kaplan, MD, an associate professor of medicine at the University of North Carolina, Chapel Hill.

The process of the protease becoming active and cleaving happens as the virus assembles, he adds. "Most of the proteins that make up the core of the particle are translated, made, and part of the precursor," Kaplan explains.

The cells are infected and taken over as a virus factory, and then a piece of the virus assembles at the membrane and buds out from the surface of the cell, which is the process of viral assembly, he says.

"If the protease does not do this job, then the virus particles are produced from infected cells, but these are not infectious, and that’s the basis of protease inhibitors," Kaplan adds. "The protease itself is embedded within this precursor, so one of the earliest steps is that the protease embedded within the precursor needs to become activated, and once it becomes activated, it can make these cleavages or cuts as the precursor liberates the mature protein."

Two of the precursors need to come together for the protease region to find itself and become active, he points out. "Now, almost all of the work on protease has been on the mature protease after it has become liberated from the precursor," Kaplan says. "We study activity within the precursor because this activity is a cascade."

Since the process is a cascade, anything that can be done to interrupt an early step in the cascade will have its effect magnified throughout the whole process of the protease doing its cleaving, he explains.

"If you interrupt it early, then you can prevent that exponential increase in protease activity that is required," Kaplan says.

The work Kaplan and a few others are doing has focused on finding ways to disrupt those early interactions, particularly looking at ways to interrupt the interaction of the two precursors, which would in turn disrupt protease interaction and kill the virus.

"We have designed a high throughput assay to look at large collections of different chemicals called compounds," Kaplan says. "These large collections are called libraries of compounds, and you can take a large library of maybe 100,000 and look at every single compound to see if it has an activity you’re interested in."

Researchers already have generated large libraries of compounds, but the next big challenge is to find a way to screen these compounds quickly, he says. "So there’s a lot of interest right now in developing an assay or test that can very rapidly screen 100,000 compounds to find ones that have some promise," Kaplan explains.

"What we’ve done is we have applied for patent protection for the development of an assay that determines whether or not two precursors can come together effectively or not and activate the protease," he adds. With this system, investigators can screen thousands of compounds a day and quickly learn whether the protease activates, he points out.

So far, Kaplan’s and co-investigator’s research indicates that it requires high levels of HIV-1 protease inhibitors to inhibit the earliest steps of precursor processing.1

Reference 

1. Kaplan AH, Pettit SC, Everitt L, et al. Inhibition of the HIV-1 protease within the GagPol precursor: implications for precursor processing, protease activation, and treatment. Presented at the IDSA conference. Boston; September/ October 2004. Poster: 740.