HARVARD COLLEGE, PRESIDENT & FELLOWS OF
Grant: $242,327 - National Institutes of Health - Sep. 9, 2009
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Award Description: This project is designed to explore a novel approach to seeking new classes of antibiotics against the tuberculosis bacterium, Mycobacterium tuberculosis, and new classes of blood thinners for humans that could be more effective than the most common anticoagulant in use today warfarin (Coumadin). We have discovered that a bacterial protein in Mycobacterium tuberculosis, that is very similar (a homologue) to vitamin K epoxide reductase (VKOR) in humans, behaves very much like and can replace one of the proteins of disulfide bond formation, a step in protein folding, in E. coli. VKOR is an enzyme involved in blood clotting in humans and is inhibited by warfarin. The Mycobacterium tuberculosis VKOR is important for the growth of the bacteria and is also inhibited by the human blood anti-coagulant warfarin; warfarin-resistant mutants of the bacterial protein look similar to those found among humans who require higher dosages of warfarin. We have a very sensitive colorimetric assay for inhibitors of Mycobacterium tuberculosis VKOR in E, coli which assesses the efficiency of disulfide bond formation via a hybrid (?-galactosidase) protein that is inactivated by disulfide bond formation. With this colorimetric assay and live E. coli cells, we can use high throughput screening to test thousands of compounds for their ability to inhibit the bacterial enzyme and those that do will then be tested both for the killing of Mycobacterium tuberculosis and in in vitro assays for inhibition of the human VKOR. The assay is such that the bacteria are colorless when the VKOR is not inhibited, but turn a deep blue color in the presence of an inhibitor of the enzyme. The recent period has been developmental in which we have found that we can use 384-well plates to do the screening. With Harvard?s Institute for Chemistry and Chemical Biology (ICCB) collection of hundreds of thousands of compounds, we will screen for inhibitors of the Mycobacterium tuberculosis VKOR homologue. In addition, we have received 594 compounds from a doctor at the NIH that have been shown to inhibit the growth of Mycobacterium tuberculosis. A doctor at ICCB laboratory will examine any of the ICCB?s compounds that pass our screen for inhibition of the growth of Mycobacterium tuberculosis and finally another doctor's laboratory will test them in a biochemical assay for inhibition of human VKOR enzymatic activity. In addition, we will attempt to get the human VKOR itself functionally expressed in E. coli, which, if successful, could allow direct screening in E. coli for potential anti-coagulants. Candidates for antibiotics against tuberculosis and for new types of anti-coagulants will assayed for their efficiency in these two processes. Collaboration with chemists in the ICCB or at Harvard University will be initiated to chemically modify any good candidates obtained in the hopes of finding drugs that are effective in one or both processes.
Project Description: We seek, by a single method, drugs to 1)kill antibiotic-resistant bacteria and 2)prevent human blood-clotting, based on the similarity of a tuberculosis protein and a human blood-clotting protein.
Jobs Summary: none yet (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on Sep. 9, 2009. Help expand these official descriptions using the wiki below.
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