This proposal is focused on the structure and mechanism of the protein prenyltransferases: farnesyltransferase (FTase) and geranylgeranyltransferase type-I (GGTase-I). These enzymes catalyze the essential addition of an isoprenoid lipid (prenylation) to over 100 proteins involved in cell growth and proliferation including members of the Ras GTPase superfamily. Inhibition of human prenyltransferases has proven to be an important target for new cancer therapies. Protein farnesyltransferase inhibitors (FTIs) are being evaluated in Phase II/III clinical trials for the treatment of cancer. Protein prenyltransferases also have been characterized from parasites and fungi that cause human disease. FTase and GGTase-I inhibitors (GTIs) show promise for treatment of parasitic infections (malaria, Chagas disease, African sleeping sickness, Leishmania), and fungal infections that are life-threatening in many immunocompromised patients with AIDS. This proposal focuses on understanding the fundamental mechanism of action and substrate specificities of the human and pathogen enzymes. We will combine X-ray crystallographic results with site-directed mutagenesis, biochemical and kinetic analyses to define the mechanism of mammalian FTase and GGTase-I reactions. Emphasis will be placed on understanding the determinants of protein and isoprenoid specificity and identifying additional intermediates in the reaction pathway. The major effort in the new project period will be to determine the crystal structures of CaaX prenyltransferases from medically important human pathogens including Trypanosomatids and fungal pathogens. Crystallographic analysis of clinically important inhibitors bound to mammalian, parasitic, and fungal prenyltransferases will be pursued. These structures are expected to facilitate drug development efforts towards highly specific FTIs (and GTIs) and provide insight for development of inhibitors to human pathogens.
