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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37GM052382-09
Application #
6923208
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
1995-05-24
Project End
2010-02-28
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
9
Fiscal Year
2005
Total Cost
$339,910
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Mabanglo, Mark F; Hast, Michael A; Lubock, Nathan B et al. (2014) Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design. Protein Sci 23:289-301
Wang, Yen-Chih; Dozier, Jonathan K; Beese, Lorena S et al. (2014) Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues. ACS Chem Biol 9:1726-35
Hast, Michael A; Nichols, Connie B; Armstrong, Stephanie M et al. (2011) Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens. J Biol Chem 286:35149-62
Fletcher, Steven; Keaney, Erin Pusateri; Cummings, Christopher G et al. (2010) Structure-based design and synthesis of potent, ethylenediamine-based, mammalian farnesyltransferase inhibitors as anticancer agents. J Med Chem 53:6867-88
Hast, Michael A; Fletcher, Steven; Cummings, Christopher G et al. (2009) Structural basis for binding and selectivity of antimalarial and anticancer ethylenediamine inhibitors to protein farnesyltransferase. Chem Biol 16:181-92
Eastman, Richard T; White, John; Hucke, Oliver et al. (2007) Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase. Mol Biochem Parasitol 152:66-71