Chagas Disease Has Been NeglectedChagas disease is regarded as a neglected disease because it mainly strikes poor people living in rural areas of Latin America, and drug companies have not viewed targeting T. cruzi as a moneymaking enterprise. But from a public health perspective, in countries such as Brazil, a focus on T. cruzi would be right on the money. In fact, for years, the life cycle of T. cruzi was depicted on the 10,000 cruzado note – with illustrations of the single-celled parasite with its whip-like tail and of the transmission of the disease via the kissing bug’s droppings. Two drugs originally developed by pharmaceutical companies to treat fungal infections – posaconazole, branded as Noxafil by Schering-Plough, and ravuconazole – are now going to be evaluated in clinical trials for Chagas disease. However, development of the two drugs was not the result of deliberately targeting key proteins in pathogens, Podust says, even though they do inhibit CYP51. Through random screening of chemical libraries, they were shown to be effective in fighting fungal infections.
Chagas Disease Drug Target Proves to Be Prize-WinningPodust recently won an annual competition among students, postdoctoral fellows and faculty from Bay Area and San Diego universities that was organized by the Wheeler Center for Emerging and Neglected Diseases (CEND) at UC Berkeley. Podust won the prize for work demonstrating that T. cruzi CYP51 was the best drug target in a neglected disease among those submitted for the contest. According to Tom Alber, PhD, CEND faculty director, “The big motivation for the contest is to educate the infectious disease research community about the characteristics of a great drug target.” Three finalists, including Podust, made their pitch from the podium. During her talk, Podust “made a powerful case” that impressed the judges from Bay Area investment and biotech companies, Alber says. First, Podust selected a target that is absolutely needed by the parasite. She then developed a high-throughput test, an assay to screen hundreds of thousands of chemicals to see which can interfere with CYP51 enzyme activity. Podust identified tightly binding inhibitor molecules through this screening, and began using one as a scaffold, modifying it with other small chemical groups to further improve the specificity of drug prototypes for the targeted enzyme. The best molecule built from this scaffold killed T. cruzi in laboratory cell cultures. More recently, Podust’s lab group has shown that this T. cruzi CYP51 inhibitor eradicates the parasite in mice.
The kissing bug, Rhodinus prolixus, spreads the parasite that causes Chagas disease. Credit: Erwin Huebner, University of Manitoba
Identifying the Three-Dimensional Structures of Drugs and Drug TargetsPodust, who for the past 10 years has been working on drug targets as a university scientist, favors drug design based on an understanding of the target’s shape. Her approach has been to precisely determine the three-dimensional structure of an enzyme that the parasite needs to survive. Enzymes bind to other molecules to chemically alter them, using chemical energy to drive crucial biochemical processes. The part of the enzyme structure that interacts with and changes the molecule to which it binds is called the active site. In targeted drug development, researchers often aim to make molecules – drug prototypes – that interfere with the enzyme’s ability to bind to or transform the natural molecule. Podust’s main expertise is in X-ray crystallography, a technique for determining protein structure by purifying the protein and getting it to form crystals. X-ray crystallographers bounce X-rays off the regularly repeating protein structures. The patterns formed by the X-rays as they are refracted off the crystal and onto a detector can be analyzed to determine the three-dimensional structures of proteins in atomic detail. Podust was the first to determine the structure of T. cruzi CYP51, and recently she determined the structure of the enzyme bound to the antifungal inhibitors, shedding light on drug action and drug resistance.
Collaborators in Their Spare TimePodust has some Sandler Center funding, and the prize for ranking first in the CEND competition included support from CEND and the UCSF Small Molecule Discovery Center (SMDC) to screen CYP51 against the SMDC’s chemical library. But much of Podust’s ongoing work for the past 10 years has been done with minimal funding and in “her spare time,” she says. “We are going to continue to explore active-site interactions with different molecules,” Podust adds. “This project requires a lot of different kinds of expertise,” she says. Despite the dearth of funds, Podust has engaged researchers from UCSF and Scripps Research Institute to continue work. She is taking advantage of the core laboratory services at the California Institute for Quantitative Biosciences (QB3), including the SMDC, and at the Protein Crystallography Beamline at Lawrence Berkeley National Laboratory. In addition, Podust says, “At UCSF, we have had more success working with parasites in animal models and cell cultures. It’s a great place to be for this project.”
A Nonazole CYP51 Inhibitor Cures Chagas Disease in a Mouse Model of Acute Infection
Patricia S. Doyle, Chiung-Kuang Chen, Jonathan B. Johnston, Stephanie D. Hopkins, Siegfried S. F. Leung, Matthew P. Jacobson, Juan C. Engel, James H. McKerrow
and Larissa M. Podust
Antimicrobial Agents and Chemotherapy