Author ORCID Identifier
https://orcid.org/0000-0001-5056-2632
Date of Graduation
12-2020
Document Type
Thesis (MS)
Program Affiliation
Biomedical Sciences
Degree Name
Masters of Science (MS)
Advisor/Committee Chair
Nayun Kim
Committee Member
Heidi Kaplan
Committee Member
Anna Konovalova
Committee Member
Ziyin Li
Committee Member
Michael Lorenz
Abstract
Trypanosoma brucei is a parasitic eukaryote and is the causal agent of the disease known as African sleeping sickness, transmitted via the bite of a tsetse fly. If left untreated, the parasite ultimately crosses the blood-brain eventually leading to death. Currently there are 5 approved drugs used to treat it, each with toxic side effects and effective at specific disease stages. A more effective and less toxic drug candidate is highly sought after. The essential enzyme, dUTPase, is an excellent drug target in the parasite while utilizing S. cerevisiae as a model system.
dUTPase is essential in T. brucei (tbdUTPase), yeast (yDut1), and humans (hDUT). dUTPase being a dimer in T. brucei but a trimer in yeast and humans, we hypothesize there may be a molecule that inhibits the function of tbdUTPase but not of yDut1 and hDUT. The endogenous yDut1 was knocked down via auxin inducible degron system in order to complement its loss by transforming plasmids containing human and T. brucei dUTPase genes. Induced expression of hDUT and tbdUTPase did not seem to complement the loss of yDut1. As an alternative strategy, the dUTPase genes from human and T. brucei were next integrated into the yeast genome at the inactive HO locus. The results of this experiment were inconclusive and remains to be further explored.
In order to identify a small molecule ligand of T. brucei dUTPase, a high throughput virtual drug screen was performed through Schrödinger’s Suite. This led to the discovery of 13 molecules that bind to tbdUTPase but not to hDUT. Out of the 13, one molecule showed cytotoxic effects against the parasite but not against yeast.
To see if the single molecule found was dUTPase specific, yDut1, hDUT, and tbdUTPase proteins were purified and their enzymatic activity were measured in the presence of the single molecule. The results indicated that the molecule found to have cytotoxic effect against T. bruceicells was not dUTPase specific. Although the results do not support the initial hypothesis, the dUTPase pathway as well as virtual drug screening hold promise and require further research.
Keywords
African sleeping sickness, human African trypanosomiasis, Trypanosoma brucei, dUTPase, virtual drug screen