Louisiana State Research Shows Drug Blocks Enzyme That Activates Bacterial And Viral Toxins

Bacteria produce a number of toxins which rapidly enter and kill cells. In anthrax, the lethal factor toxin must bind to another part of the anthrax toxin, called the PA molecule, before it can enter and destroy a cell. But before binding can occur, the PA molecule produced by the bacteria must be made smaller. Furin, a protein-cutting enzyme or protease, which sits on the outside of cells, cuts the PA molecule, making it small enough for the lethal factor toxin to attach. Lethal factor toxin cannot bind to PA that has not already been cut by furin; therefore, without cut PA, lethal factor toxin loses the ability to bind to and enter the cell, and becomes harmless.

Working on the theory that if the action of furin could be blocked, the toxins would not be activated and therefore unable to kill cells, the research team set out to make a peptide that would suppress furin activity. In collaboration with a research group in California (Torrey Pines Institute for Molecular Studies), the LSUHSC group developed the furin inhibitor, D6R, for which a patent application has now been filed. The LSUHSC research group under the direction of Dr. Iris Lindberg, Professor of Biochemistry, included current postdoctoral fellow Dr. Miroslav S. Sarac, and past fellow Dr. Angus Cameron.

In the current paper, the LSUHSC research scientists examined the therapeutic potential of D6R against Pseudomonas aeruginosa exotoxin, both in cell culture and in live animals (mice). Various concentrations of D6R were tested, with no apparent side effects, regardless of dose. (However, no tissue microhistology has yet been done, and these studies are needed to reveal any subtle damaging effects.) The survival rate of cells in culture 48 hours after treatment with D6R–given at the same time as Pseudomonas toxin-- was significant–50%. The survival rate of the group of mice treated with D6R for two days prior to administration of toxin was also about 50% at seven days. The group of mice treated with D6R at the same time toxin was administered showed a 25% survival rate.

Pseudomonas aeruginosa is an opportunistic pathogen causing urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia and a variety of systemic infections, especially in those with immunosuppression related to cancer, HIV and severe burns. P. aeruginosa is known for its resistance to antibiotics. Although some antibiotics are effective against some strains, even those antibiotics are not effective against all strains.

Sepsis, a systemic infection, is the leading cause of death among critically ill patients in the US. It is estimated that about 750,000 American people develop sepsis each year and that more than 200,000 die from it. One of the factors contributing to the high mortality of sepsis (which has increased more than 90% in the last 20 years according to the National Vital Statistics Report, 2000,) is damage from excessive production of cytokines (chemical messengers of the immune system). The LSUHSC scientists found that D6R was not only able to protect cells from lethal toxins, but to do so without invoking a cytokine response itself. D6R also dramatically lowered the production of one cytokine, TNF.

According to the researchers, D6R appears to exhibit a number of potentially promising attributes. It is able to cross cell membranes; it is small enough to achieve useful therapeutic concentrations; and it exhibits no apparent toxicity at concentrations 100x therapeutic levels. It could also prove useful in treating infection from other viruses and bacteria whose toxins are dependent upon furin activity for activation. Besides Pseudomonas and anthrax, these include Ebola, clostridium, diphtheria, shiga, RSV, HIV-1, infectious bronchitis and yellow fever among others.

Dr. Lindberg was recently awarded a grant by the National Institute of Allergy and Infectious Diseases to test D6R against anthrax toxin in both cells as well as animal models (rats and mice). She will further examine the structural requirements of D6R-related molecules against furin with the idea of eventually using D6R (or a better drug developed through these studies) as a therapeutic drug for anthrax, Pseudomonas and other furin-dependent infections. She will also conduct safety and toxicity studies during the grant period.

"This is very exciting research," said Dr. Lindberg. "We hope that D6R-like furin inhibitors will represent effective and potent new drugs that will not only prove a viable approach to combat some types of bacterial infection, but also add to our country's bioweapons defense arsenal."