HTP Screen of Compounds that Inhibit Giardia Fructose-1,6-Bisphosphate Aldolase

Giardia lamblia is a human pathogen afflicting impoverished nations, and the most common cause of outbreaks of diarrhea in the United States. Giardia has been classified by the CDC as a category B bioterrorism organism. Giardiasis is treated with metronidazole or tinidazole, compounds that have undesirable side effects. Moreover, increasing resistance to drug regimes and recurrence are a concern. It is thus clear that alternative drug treatments are needed. <P> We have identified the G. lamblia Class II fructose-1,6-bisphosphate aldolase (FBPA) as an excellent candidate for drug development and thoroughly characterized its kinetics, catalytic mechanism (requiring Zn2+ cofactor for electrophilic catalysis), and crystal structure. FBPA, a key glycolytic pathway enzyme, is the sole aldolase in Giardia. As the organism requires glucose for growth, FBPA is crucial for its survival. The human FBPA belongs to the Class I aldolases that have a distinctly different substrate binding site structure and a radically different catalytic mechanism (proceeding via a lysine-Schiff base intermediate). Thus, the probability of identifying an inhibitor via high throughput compound screening that impedes the glycolytic pathway in the pathogen but not in the human host is considered to be very high. The G. lamblia FBPA catalytic activity will be assayed using several different assays, which in combination will identify potential specific inhibitors and eliminate 'false positives'. After filtering compounds with undesirable characteristics, the potential inhibitors will be evaluated to determine the type of inhibition and the inhibition constants. The best inhibitors will be examined for growth inhibition of Giardia trophozoites and cytotoxicity towards human cells. Synthetic chemistry will be employed to improve potential inhibitors, integrating SAR studies with crystal structure determination of enzyme/inhibitor complexes.<P> PUBLIC HEALTH RELEVANCE: Giardia lamblia is a human pathogen afflicting billions of people annually and a category B bioterrorism organism. Giardiasis is treated by drugs that have undesirable side effects and that enable recurrence and increasing resistance. To facilitate discovery of better drugs, we have identified the G. lamblia Class II fructose- 1,6-bisphosphate aldolase as an excellent candidate for drug development because it is an essential enzyme for the survival of the organism and it functions by a different mechanism than its human counterpart. Here we propose a high throughput screening of the NIH compound library to identify and characterize new selective inhibitors of the Giardia fructose-1,6-bisphosphate aldolase.