Duke Researchers Identify The Mechanisms By Which Drug Combinations Kill Fungal Infections

Led by Dr. Joseph Heitman, who is a Howard Hughes Medical Institute investigator at Duke, the research team explored the mechanism behind the effectiveness of administering the azole class of antifungal drugs such as fluconazole (Diflucanâ), with cyclosporin or FK506, two drugs commonly used to suppress rejection of transplanted organs and tissues.

The study is published in the Feb. 15 issue of the EMBO Journal, a journal published by the European Molecular Biology Organization.

Cyclosporin and FK506 block rejection of transplanted organs by inhibiting immune-system signaling pathways that activate T-cells. These drugs block calcineurin, an enzyme that plays a critical role in activating immune cells. Suppression of the immune system is necessary for transplanted organs to survive, but renders patients susceptible to infections by bacteria, viruses and fungi.

"The azoles are very nontoxic in humans, but the problem is they don't kill fungal cells. Instead, these drugs act by inhibiting fungal cell growth. The cells stop growing, but they don't die. As a result, a lot of fungal isolates become resistant to the azoles," Heitman said.

The study was funded by grants from the National Institute of Allergy and Infectious Diseases.

Unlike bacteria or viruses, fungi are eukaryotic cells that resemble cells of the human body. Fungal infections can thus be difficult to treat, Heitman said. The majority of transplant recipients suffer one or more infections and these infections are a significant cause of morbidity and mortality. Cryptococcal infections occur to two to three out of every 100 transplant recipients and is associated with a 50 percent mortality rate. Candida albicans, which can cause thrush, esophagitis or vaginitis, is the most common fungal infection and is common in hospital settings.

Heitman said these findings open the door to the clinical use of drug combinations and could lead to the identification of additional drug targets.

"There's an ongoing need to develop better antifungal drugs. There is an increasing population of people who are at risk and some infections prove very difficult to treat," Heitman said.

"There are relatively few drugs to treat these infections, some have serious side effects, and drug resistant isolates have emerged. For some fungal infections, such as aspergillus infections in the lungs and cryptococcal infections in the brain, there is a 50 percent risk of death. These studies reveal new ways in which existing drugs can be combined to combat fungal infections and improve therapy," he said.

Combining fluconazole with either cyclosporin or FK506 was previously found by other researchers to potently kill fungal cells in the test tube, and had been proposed as an approach to combat difficult to treat fungal infections.

The multi-drug antifungal treatment concept was tested in mice in 2000 by a research team led by Dominque Sanglard, an investigator at the CHUV Hospital in Lausanne, Switerzerland, and an international scholar of the Howard Hughes Medical Institute. Their studies demonstrated that the combination of cyclosporin and fluconazole successfully eradicated fungal infections, but the cellular targets and the mechanism of drug action were not understood. Elucidating the molecular targets could open the door for new or improved drugs, Heitman said.

"The big question was, ‘What are the molecules that cyclosporin and FK506 target to allow the azole drugs to kill the fungal cell?' One previous proposal was that cyclosporin inhibits the pumps that extrude the fluconazole drug from the cell. We found that this is not the mechanism of action. Instead, both cyclosporin and FK506 enter the fungal cell and inhibit calcineurin, which is their well-established target.

"We discovered that calcineurin is a component of a fungal stress response that allows the cell to survive assault on the membrane by the azoles. Inhibition of calcineurin cripples this stress response and now allows the azoles to kill rather than simply maim the fungal cells," he said.

The researchers at Duke, which also included Maria Elena Cardenas and John McCusker from the departments of genetics and microbiology, and Dr. John Perfect from the department of medicine, used molecular genetic approaches to pinpoint calcineurin as the target of the synergistic drug dynamic duo.

When a mutation was introduced into calcineurin that prevents binding to FK506, this conferred resistance to the drug combination. They also made loss of function mutations in which they removed calcineurin from the cell. The cells were viable, but they now died when exposed to azole drugs that fail to kill wild-type cells. Thus, these studies reveal new ways in which existing drugs can be combined to combat fungal infections and improve therapy.