March 23, 2001

Protein structure to aid antifungal drug design

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Michael Waterman and Larissa Podust display a model of CYP51, a protein targeted by antifungal drugs. Their findings are published in the March 13 Proceedings of the National Academy of Sciences. (photo by Dana Johnson)

Protein structure to aid antifungal drug design

Athlete’s foot, beware! Yeast infections, look out! An army of powerful new drug weapons may soon be dispatched to wipe you out.

That is the prediction of Vanderbilt University Medical Center researchers who recently reported the structure of a key protein targeted by antifungal drugs. Their findings, published March 13 in the Proceedings of the National Academy of Sciences, provide the tools for improved antifungal drug design.

Larissa M. Podust, Ph.D., research instructor in Biochemistry, and Michael R. Waterman, Ph.D., Natalie Overall Warren Distinguished Professor and Chair of Biochemistry, studied an enzyme called CYP51. CYP51-present in fungi, plants, and animals-is essential to the synthesis of sterols, such as cholesterol in animals. Its activity is required by fungi, and the widely used “triazole” class of antifungal drugs work by binding to CYP51 and blocking its action.

While these drugs have had a major impact on the treatment of both topical and systemic fungal infections, there is a need for new and improved drugs, Podust said. Because the available drugs can block both the fungal and human versions of CYP51, they can cause undesirable side effects. And resistance to the current drugs is becoming an increasing clinical problem as wily fungi acquire mutations that let them survive, even in the presence of antifungal drugs.

The various CYP51 enzymes-from fungi, plants, and animals-have been extensively studied, Podust said, but because they associate with cell membranes and are “insoluble,” it has been impossible to study their structures using a technique called X-ray crystallography.

Determining the structure of a protein is akin to taking a molecular snapshot and being able to “see” the protein’s features-the bumps and dents that influence substrate and drug binding. Drug developers can use the information to design drugs that “fit” into the protein, like a key into a lock.

A lucky break came to investigators studying CYP51 when a new member of the protein family, a CYP51 from Mycobacterium tuberculosis, was found to be soluble and amenable to X-ray crystallography.

“It was clear that the structure of this new CYP51 had to be solved,” Podust said, “and we were successful in doing that.”

“It makes me very proud that the structure was completely determined using the department of Biochemistry’s X-ray crystallography laboratory,” Waterman said.

Even though the structure they reported is for a bacterial CYP51, the results will be quickly applied to the fungal, plant, and human versions of the protein, Podust said.

“It is a well established approach to use one protein structure to model the structure of a similar protein that cannot immediately be studied by X-ray crystallography,” she said. “The CYP51 structure we determined will be used like a template, and a computer program will fit the fungal and human sequences to this template and conclude how the binding sites look.”

Drugs designed to fit into the fungal CYP51 binding site-but not the human binding site-would theoretically be free of side effects, Podust said. “And it is possible that drugs designed to fit the bacterial CYP51 binding site will be important to the treatment of tuberculosis.”

The structure will also be useful in addressing the issue of drug resistance, she said. Immunosuppressed patients subjected to long-term antifungal drug therapy are especially susceptible to resistant strains of the fungus Candida albicans. Other investigators have isolated resistant strains and determined mutations in the CYP51 protein that correlate with drug resistance.

Podust and Waterman mapped the location of these mutations in their CYP51 structure and found “an interesting distribution of mutations,” Podust said. “We didn’t find any mutations that affect the drug binding site, which is unusual. Instead, the mutations affect areas of the protein involved in conformational changes that might happen during steps of the catalytic cycle.”

In addition to the implications of the current findings for antifungal drug design, they also shed light on a more basic question of how the CYP51 enzymes accomplish their normal job during sterol synthesis.

CYP51 proteins belong to a large superfamily of P450 enzymes. The 1500 known P450 enzymes have roles in synthesizing molecules (like CYP51 does) and in detoxifying chemicals from the environment.

The CYP51 structure is only the eighth P450 structure to be solved, and it is the first for a P450 enzyme involved in a mammalian biosynthetic pathway, Podust said. Surprising aspects of the structure suggest new mechanisms for how the enzyme works.

“The structure has interesting new features compared to other P450 structures seen so far,” Podust said. “It gives us the opportunity to think about new mechanisms for catalysis and how these enzymes evolved.”

In addition to modeling the fungal and human forms of CYP51, the investigators have initiated efforts to crystallize human CYP51. “I am optimistic that the structure of the human enzyme will be determined within the next year or two,” Waterman said.

Thomas L. Poulos, Ph.D., professor of Molecular Biology and Biochemistry at the University of California, Irvine collaborated on the studies. The research was supported by the National Institutes of Health.