St. Louis, Jan. 5, 1999 — Like a man strapped to a pack of dynamite, HIV-infected cells will self-destruct when rigged with a lethal protein combination. The virus lights the fuse when it tries to reproduce.
The study, reported today in the January issue of Nature Medicine, makes use of a new technology for introducing large proteins into cells – a long-held dream of the pharmaceutical industry.
"This Trojan horse approach should be applicable to many other infectious diseases, such as hepatitis C, malaria and herpes," says Steven F. Dowdy, Ph.D., who led the research. "We also hope that future modifications will allow us to selectively kill cancer cells."
Dowdy is an assistant investigator of the Howard Hughes Medical Institute and an assistant professor of pathology and medicine at Washington University School of Medicine in St. Louis. Adita M. Vocero-Akbani, Ph.D., a research associate of the Howard Hughes Medical Institute, is the paper's lead author. The work was performed in collaboration with Nancy Vander Heyden, a research associate in the laboratory of Lee Ratner, M.D., Ph.D., professor of medicine, molecular microbiology and pathology.
"This novel method of introducing cytotoxic proteins into HIV-1-infected cells is a therapeutic approach that could complement antiviral drugs that are currently in clinical use," Ratner says.
The virus that causes AIDS uses a scissors-like protein called a protease to cut out enzymes it needs for reproduction. Drugs called protease inhibitors, which are extending many lives, sit in the hinge of the scissors, preventing them from doing their job. But mutations in the protease can make the drugs ineffective. The drugs also inhibit a patient’s own proteases, so they often are toxic.
Riding his bike to work one day, Dowdy thought, "Why not use the viral protease to kill the cell instead of using a drug to inactivate the protease?"
To accomplish this, Vocero-Akbani pieced together a novel protein. First she took a protein that can slip through cell membranes. Then she attached two pieces of a human enzyme called caspase-3, which, when activated, enables cells to commit suicide – this enzyme normally is kept under lock and key. She joined the three pieces together with cleavage sites from HIV that tell the viral protease where to cut. She then exposed cultured HIV-infected cells to this fusion protein, which was smuggled into all of the cells by its protein transduction motif – the protein that travels through cell membranes.
Because the cells contained actively reproducing HIV, they also contained the viral protease. This enzyme chopped up the fusion protein at the "Cut here" sites, freeing the two pieces of caspase protein. Acting in tandem, these pieces triggered a chain of suicidal events. Within a few hours, cells infected with active HIV had killed themselves. Uninfected cells were spared because they contained no viral protease to activate the caspase.
Dowdy envisions giving the fusion protein to patients as an aerosol that would attach to the membranes of the lungs and then move into the bloodstream. It would travel through tissue by moving from cell to cell.
"The beauty of the protein fusion approach is that it is unlikely to be sabotaged by viral mutations," he says. "HIV uses its protease to cut up a gigantic protein at eight to 10 sites. Mutating all eight to 10 sites in this polyprotein at the same time would be statistically impossible. The virus also would have to change the specificity of the protease at the same time."
The fact that several HIV strains already exist shouldn’t be a problem. "You simply adapt the killing molecule by inserting the cleavage sites from those different HIV strains," Dowdy says.
His group described its success in getting large proteins into cells in the December issue of Nature Medicine. Although scientists previously had demonstrated that the protein transduction motif could be used for this purpose, they were unable to introduce sufficient amounts of proteins to affect the functions of cells. On another bike ride, Dowdy thought that proteins might be easier to introduce if they were unfolded first. "Denatured proteins seem to transduce more efficiently across the cell membrane and appear to be more easily refolded inside the cell than partly misfolded proteins," he says.
By genetically manipulating bacteria, Dowdy’s group has made 60 transducible proteins ranging in size up to 110,000 daltons. In contrast, typical drugs are smaller than 800 daltons — "like the point of a pen compared with a fist," Dowdy says.
When small drugs interact with cellular targets, they attach only to one part of the protein’s surface and often latch onto several other proteins nonspecifically. Larger proteins fit only onto the molecules for which they were designed, could be given in substantially lower doses and therefore should cause fewer side effects. Potentially useful proteins occur within our own bodies as well as in other living organisms. "By using these transducible proteins as drugs, you could take advantage of 5 billion years of evolution," Dowdy says. "This work describes the first example of an entirely new field of protein therapy."
He believes fusion proteins could be designed to combat many infectious organisms that use a protease during their lives in humans. He also is developing fusion proteins that hopefully will kill prostate cancer cells. Removing prostate cells one by one is unlikely to cause the side effects of current treatments for prostate cancer, Dowdy says.
The research was supported by the National Institutes of Health and the Howard Hughes Medical Institute.
The technology from Dowdy's laboratory has been licensed to IDUN Pharmaceuticals of La Jolla, Calif., and Life Technologies of Rockville, Md.
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