Advisor
Keenan, Susan M.
Committee Member
Dong, Aichun
Committee Member
Schountz, Tony
Committee Member
Mackessy, Stephen P.
Department
Biological Education
Institution
University of Northern Colorado
Type of Resources
Text
Place of Publication
Greeley (Colo.)
Publisher
University of Northern Colorado
Date Created
8-1-2013
Genre
Thesis
Extent
192 pages
Digital Origin
Born digital
Description
The protozoan parasite, Plasmodium (P.) falciparum, causes the most virulent form of malaria and is a significant source of mortality in the developing world. Increasing resistance of this parasite to traditional antimalarials has necessitated the identification of new targets for antimalarial drug design. Protein kinases, which mediate critical cellular processes such as proliferation, growth, and apoptosis, make attractive drug targets as the deregulation of cellular phosphorylation events has been linked to human diseases. In recent years, kinase inhibitors have successfully treated such diseases as chronic mylogenous leukemia and renal cell carcinoma. The unique structural and mechanistic aspects of many P. falciparum kinases make these proteins potentially useful drug targets and the inhibition of these macromolecules provides a means to examine heretofore undescribed cellular mechanisms within these dynamic parasitic organisms. First, three libraries of known kinase inhibitors were screened against the malaria kinase, PfPK7, using a ATP luminescence assay to determine which of these molecules had affinity for the ATP binding site of this kinase. Computational methods were employed to determine how small molecule inhibitors were binding to the PfPK7 ATP-binding site and properties that are important for determining the druglikeness of each compound were predicted using commercially available software. In addition, small molecules were screened against P. falciparum strain W2 to determine their ability to inhibit intraerythrocytic stage growth using a SYBR Green I growth assay. Finally, the well characterized kinase inhibitor Purvalanol B was applied blood stage cultures of P. falciparum strain W2 and differences in protein expression between inhibitor-treated and wildtype parasites were determined using a shotgun proteomics approach. Out of the 244 small molecules tested, eight were found to have affinity for the ATP-binding site of PfPK7 and the probable interactions that contribute to small molecule binding as well as the predicted properties that contribute to druglikeness were described. While several small molecules were able to inhibit intraerythrocytic parasite growth, most inhibitors of PfPK7 identified in the luminescence screen did not significantly inhibit P. falciparum growth Disruption of parasite development with the kinase inhibitor Purvalanol B yielded differences in protein expression between wildtype and inhibitor-treated parasites. These data further characterize the orphan kinase PfPK7 and may suggest this target may not be ideal for antimalarial drug design as its inhibition does not correspond with attenuated P. falciparum growth in blood stage cultures. Finally, protein differences resulting from kinase inhibitor treatment during parasite cell cycle aid in the elucidation of the heretofore poorly understood P. falciparum cell cycle mechanisms.
Degree type
PhD
Degree Name
Doctoral
Language
English
Local Identifiers
Bullard_unco_0161D_10256
Rights Statement
Copyright is held by author.
