Date of Award

5-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Nina Goodey

Committee Member

Ueli Gubler

Committee Member

John Siekierka

Abstract

Dihydrofolate reductase (DHFR) is an essential enzyme that plays an important role in the production of cofactors that are required in deoxyribonucleic acid (DNA) synthesis. The literature suggests that DHFR is a potential drug target for the elimination of the parasitic worm, B. malayi. B. malayi is one of the causative parasites of lymphatic filariasis, a globally neglected tropical disease. In this study, we expressed and purified B. malayi DHFR. Expression was carried out in E. coli with a His6-tagged construct, and purification was achieved through affinity chromatography using a special strain of E. coli cells called “LOBSTR.” The resulting purified and active enzyme was used for steady-state kinetics characterization and inhibition studies. The catalytic activity, kcat, was found to be 1.4 ± 0.1s-1, the Michaelis Menten constant, KM, 14.7 ± 3.6uM for dihydrofolate, and the equilibrium dissociation constant, KD, 22 ±0.01 uM. B. malayi DHFR was compared to 13 other DHFR homologs in terms of ligand specificity determining residues; L. major, T. cruzi, T. bruci, and T. gondii exhibited the highest homology. Known inhibitors of these DHFR homologs were assayed with B. malayi DHFR and an inhibition profile was created for the enzyme. IC50 values were determined to be 0.0036 ± 0.0008 uM for methotrexate, 109 ± 34 uM for pyrimethamine, 32 ± 22 uM for trimethoprim, 771 ± 44 uM for cycloguanil, >20,000 pM for 2,4- diaminopyrimidine, and 154 ± 46 uM for 2,4-diaminoquinazoline. These results provide the basis for the development of more potent and less toxic compounds that can inhibit B. malayi DHFR and help cure lymphatic filariasis.

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