Medicinal Chemistry, Faculty of Pharmacy
University of Manitoba
Winnipeg, Manitoba, Canada R3E 0T5
Prevention of Oxygen Radical Damage and Targeting Topoisomerase II
The various research projects in this laboratory aim at:
- an understanding of the mechanisms of oxygen and nitrogen free radical tissue damage and its prevention by antioxidant drugs, and
- an understanding of the mechanism of action of topoisomerase II and the anticancer drugs that target this enzyme.
Oxygen free radical damage has been implicated in various drug toxicities, ischemic-reperfusion damage such as that occurs after a stroke or heart attack, the aging process, radiation injury, inflammation, and in a variety of other conditions and diseases. A number of recent studies have focused on the bisdioxopiperazine antioxidant drug dexrazoxane (ICRF-187), which is currently being used to prevent the dose-limiting cardiotoxicity caused by the antitumor anthracycline doxorubicin. Doxorubicin is thought to cause myocardial tissue damage through iron-mediated oxygen free radical production. The bisdioxopiperazines may be preventing iron-based free radical damage by scavenging free iron, thus preventing its participation in oxygen radical production. The bisdioxopiperazines have also been shown to be catalytic inhibitors of DNA topoisomerase II. Topoisomerase II is the target of a number of very important anticancer drugs, including doxorubicin, daunorubicin, etoposide, amsacrine, and mitoxantrone. The mechanism of action of topoisomerase II is being studied using dexrazoxane and other bisdioxopiperazines as probes.
- The molecular mechanism of the cardioprotective, antioxidant, and anti-apoptotic activity of dexrazoxane is being studied by epifluorescence microscopy in a cardiac myocyte model.
- The metabolic activation and pharmacokinetics of the bisdioxopiperazines is being studied in tissue homogenates, in hepatocyte suspensions, in cultured myocytes, and in vivo in clinical and animal studies.
- The cytotoxicity of dexrazoxane in combination with other anticancer drugs with which it is being used clinically is being studied in a cultured cell model in order to look for synergistic or antagonistic effects.
- A photoaffinity labeled etoposide analog has been synthesized. In collaboration with researchers in the Department of Physics and the University of Pittsburgh, it is being used to identify the etoposide binding site on topoisomerase II and to probe the mechanism of topoisomerase II using a mass spectrometric proteomics approach.
- A number of dexrazoxane resistant cell lines have been isolated, one of which has been shown through DNA sequencing to contain a mutant topoisomerase II. The other mutants will be sequenced to identify the dexrazoxane binding site on topoisomerase II.
- Molecular modeling is being used to design bisdioxopiperazine analogs in order to improve their antioxidant and anticancer effectiveness. Structure-activity studies of bisdioxopiperazines that inhibit topoisomerase II are being carried out to define the structural features that are necessary for bisdioxopiperazine binding to topoisomerase II.
- Using an isolated cardiac myocyte cell model the bisdioxopiperazines are being studied to see if they can prevent a variety of other free-radical based drug-induced toxicities, and if they can prevent hypoxia-reoxygenation damage such as that caused by stroke or heart attack.
- The metabolism and pharmacokinetics of the bisdioxopiperazine dexrazoxane is being studied in humans and in an animal model with a view to understanding its activity and increasing its effectiveness and extending its use to other oxidative stresses.
- The mechanism of topoisomerase II is being investigated in a cell model using dexrazoxane in combination with other anticancer drugs.
- Using molecular modeling the design, synthesis, and in vitro testing of more effective antioxidant and anticancer bisdioxopiperazines is being pursued.
- The activation of dexrazoxane to its metal chelating form in cardiac myocytes is being studied using the fluorescent iron sensing dye calcein.
- Several other iron chelating drugs is being studied to determine their ability to prevent doxorubicin-induced and hypoxia- reoxygenation oxidative stress.
Our laboratory is well equipped for a range of biological and biochemical studies. Equipment includes computer-controlled UV-vis double beam spectrophotometers, a spectrofluorimeter, fluorescence and absorbance plate readers, computers for molecular modeling and data analysis, and an HPLC apparatus with both photometric and fluorometric detection. The lab also has a new Bruker EMX electron paramagnetic resonance (EPR) spectrometer for free radical, and nitric oxide and oxygen free radical spin trapping studies. The cell culture facility in the laboratory has laminar flow hoods, carbon dioxide incubators, and color and B&W CCD deconvolution epifluorescence and phase contrast microscopes. The lab also contains other equipment for enzyme purification, cell component isolation, and gel electrophoresis equipment for Western blots and quantitative DNA gel assays. The laboratory is open to collaborations in the general area of nitrogen and oxygen free radical research and to collaborations that can make use of our equipment or knowledge base.
Two grants from the CIHR and a grant from the Canada Research Chairs program currently support our research.
Some recent publications are listed here.
For additional information, please contact:Dr. Brian Hasinoff (Canada Research Chair)
Faculty of Pharmacy
University of Manitoba
750 McDermot Avenue
Winnipeg, Manitoba R3E 0T5 Canada
Voice: (204) 474-8325 (office) or (204) 474-6760 (lab)
FAX: (204) 474-7617
WWW home page: http://home.cc.umanitoba.ca/~hasinof/~hasinof.htm