Date of Completion

Spring 5-1-2014

Thesis Advisor(s)

Amy Anderson

Honors Major

Doctor of Pharmacy

Abstract

The development of drug compounds begins with the identification of well-validated targets. Conserved in bacterial, fungal, and mammalian species, the folate biosynthetic pathway performs critical processes to promote nucleic acid synthesis and maintain cellular function. Medicinal chemists have targeted dihydrofolate reductase (DHFR), an essential enzyme in this metabolic process, for several years. In fact, anti-folates acting on this pathway have potential roles against infectious diseases. This project examines a para-substituted drug compound called UCP111E, which is directed against dangerous fungal species, like Candida albicans and Candida glabrata. Since crystal structures of C. albicans DHFR (CaDHFR) and C. glabrata DHFR (CgDHFR) with UCP111E have already been solved, the main objective is to now deduce the structure of human DHFR (huDHFR) with the drug compound.

Sample preparation procedures in this project used Qiagen EasyXtal 15-well trays to plate various conditions for crystallization. Buffer (i.e., Tris), salt (i.e., lithium sulfate), precipitant (i.e., PEG 4000), and additive (i.e., ethanol) remained constant for each well in the trays. However, non-volatile additives (i.e., 1,8-diaminooctane, strontium chloride, and calcium chloride) varied.

Steps to improve the conditions with 1,8-diaminooctane produced plate-like crystals, while strontium chloride formed hexagon-shaped crystals, and calcium chloride, crystal rods. Only crystals formed with 1,8-diaminooctane and calcium chloride were large enough to diffract. However, diffraction patterns revealed the presence of salt, rather than protein, in the structures. Future projects hope to continue optimizing conditions with strontium chloride and calcium chloride to crystallize the huDHFR/UCP111E complex.

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