OXFORD, Miss. – What began in 1984 as a contract to screen compounds for activity against opportunistic infections that threaten the lives of people with suppressed immune systems has become one of the longest continually funded antifungal research programs in the history of the National Institutes of Health.
The program’s principal investigator is the University of Mississippi’s vice chancellor for research and sponsored programs, Alice Clark, who first received funding for the work from NIH’s National Institute of Allergy and Infectious Diseases, or NIAID, 30 years ago.
At the time, a host of opportunistic infections were ravaging the bodies of people with suppressed immune systems due to AIDS, cancer chemotherapy or immune-suppressing drugs, but treatments were sorely lacking.
With that first NIH-NIAID funding, Clark, co-principal investigator Charles Hufford and others in the university’s School of Pharmacy focused on finding new drugs to treat fungal infections such as systemic candidiasis. Candidiasis is caused by Candida albicans, which produces localized yeast infections (i.e., oral thrush, vaginitis and diaper rash) that are not a problem for people with healthy immune systems. But in people with suppressed immune systems, the organism can invade the whole body and attack the organs.
During the project’s early years, Clark and her team found and patented several compounds that kill or inhibit C. albicans, and the compounds represented totally new and different classes of antifungal antibiotics. That’s because, rather than synthesizing analogs of existing drugs, which were likely to have the same toxicities and resistance problems as the parent drug, the researchers focused on isolating compounds from higher plants – trees, shrubs and flowers – as well as microorganisms, the traditional source of antifungal antibiotics.
Because of such progress, Clark and Hufford received a $1 million contract renewal in 1987 (one of only three awarded nationwide) to continue their work for five more years. Two years later, they received a grant from NIAID for similar work. With Clark as its principal investigator, that grant (RO1-AI-27094) has been renewed four times and been funded with nearly $5.9 million since 1989.
“I believe this is probably the longest antifungal research program in NIH history,” said Hufford, Clark’s spouse and the pharmacy school’s associate dean for research and graduate programs.
“It has been an honor to be supported by NIH, and through NIH by my peers in the scientific community, for 30 years,” Clark said. “I feel extremely fortunate to have had such a long and successful relationship with them. I am also grateful to Chris Lambros, my program officer at NIH, who has been extremely engaged, knowledgeable, helpful and visionary. He has been an incredible resource to my group and to the whole scientific community.”
Today, drugs such as fluconazole and caspofungin are frequently used to treat infections caused by Candida and other fungi (e.g., lung infections caused by Aspergillus and meningitis caused by the yeast Cryptococcus), but resistant strains of these pathogens are diminishing the drugs’ utility and effectiveness. Because of that, Clark and her colleagues are not only evaluating natural products for antifungal activity but also their ability to work in combination with fluconazole and caspofungin to restore their effectiveness.
With the help of pharmacy school research scientists such as Ameeta Agarwal, Xing-Cong Li and Melissa Jacob, Clark’s team has identified a small number of compounds that do just that. Some help keep fluconazole inside fungal cells, where it can do its work, while others prevent fungal cells from repairing the damage to their walls that caspofungin causes.
“Over time, microbes and other pathogens develop resistance, so it is important to continually develop new drugs that kill them or that restore the effectiveness of existing drugs in resistant strains of the pathogens,” Clark said. “We evaluate thousands of samples of plants and microorganisms from all over the world to see if they can do either or both of these things. We then isolate, purify and determine the chemical structure of the individual natural product compound most responsible for the effect and determine its mechanism of action.”
Unravelling just what makes fungal pathogens susceptible, or resistant, to drugs is the key to devising new ways to kill them and treat the infections they cause.
“From that information, we can design new biological tests to help discover other new compounds,” Clark said.
For these particular studies, the researchers use the yeast Saccharomyces cerevisiae, because of its simple genetics and biochemistry, and a host of sophisticated genomic, genetic and proteomic (analysis of structure and function of proteins and/or enzymes) technology.
“We use technology that shows how an organism’s genes respond after exposure to the natural product,” Clark said. “First, we use transcript profiling technology to identify biological pathways that respond to the candidate drug. Once a target pathway is identified, we conduct follow-up studies to pinpoint the precise drug target.”
Those follow-up studies include testing to determine whether mutant strains of yeast that lack those biological pathway genes are more sensitive to the potential drug, or to search for specific enzymes or metabolites in the target pathways, Clark said. Finally, the researchers test the effectiveness of the potential drug against fungal pathogens, using similar approaches to what they use in model organisms.
Restoring the potency of existing antifungal drugs presents multiple advantages, Clark said. “Increasing the intracellular concentration of the primary drug, for example, can lead to shorter durations of therapy, reduced dosages and fewer side effects.”
Although the goal of Clark and her team is to improve the quality of life of millions of people with immune disorders worldwide, their work is a continual cycle of concurrent and interrelated studies.
“Collaboration is the key to success of this project,” Clark said. “The work simply could not be done by any single investigator, and we benefit from collaborations with researchers in other academic institutions, government labs and companies.
“I have had the great privilege of working with many outstanding collaborators throughout my career but none more so than my UM colleagues on this project: Drs. Agarwal, Li and Jacob, who lead our efforts in molecular biology, natural products chemistry and antifungal screening, respectively.”