Aaron Ramsey & Ana Lisa Valenciano

Killer instincts: two Ph.D. students search for biological targets in the parasite that causes sleeping sickness

Two Virginia Tech graduate students are investigating gene targets in Trypanosoma brucei, a vector-borne parasite that causes sleeping sickness.

According to the World Health Organization, as the parasite spreads through the body, it causes fever, headache, and intense aches and pains. When it reaches the brain, it causes swelling, creating an intoxicated-like effect of slurred speech, confusion, and difficulty walking, followed by coma and usually--if left untreated--death.

Studying sleeping sickness is increasingly important to people who live in and travel to endemic countries in sub-Saharan Africa. About 60 million people in 36 of these countries are at risk of infection with T. brucei, which also infects livestock and devastates the region’s agricultural industry, accounting for nearly $5.5 billion lost per year from the agricultural economy. The people who suffer are generally from impoverished rural villages where the parasite’s transmitter, the tsetse fly, is pervasive.

Aaron Ramsey, a third-year Ph.D. student in the lab of Zachary Mackey, an assistant professor of biochemistry in the College of Agriculture and Life Sciences, is working to identify specific drug targets that can be exploited to kill the parasite without also causing harm to its human host.

Currently, two drugs exist to treat advanced stages of this disease but come at a cost to those infected. Melarsoprol, for example, is an arsenic derivative, meaning its side effects are equivalent to arsenic poisoning—severe fever, rash, vomiting, and encephalopathy or brain dysfunction, and in some cases death.

Eflornithine, another option for the second stage of one form of the disease, costs more than one year’s salary for many of those afflicted, and requires intravenous treatments every four hours for up to two weeks. ‘Mild’ side effects may include hair loss, lowered immune system, lack of appetite, diarrhea, and vomiting.

In an attempt to add to the list of possible therapeutic interventions, Ramsey studies an enzyme that may be responsible for repairing DNA in the parasite. In particular, Ramsey studies DNA ligase I, an enzyme that in normal eukaryotic organisms, like people, joins DNA strands back together during genome replication and repair, thus sustaining the life of the organism.

Ramsey’s goal is to identify differences in how this enzyme works in the parasite that can be exploited for drug development.

A better understanding of how this and other genes work in T. brucei is key to developing new drugs to treat the parasite, which is the main goal of Mackey’s lab. More specifically, the goal is to investigate which T. brucei genes that regulate key biological processes like DNA replication make the best drug targets.

The work of Ana Lisa Valenciano, a fourth-year Ph.D. student in the lab, focuses on characterizing a kinase that regulates DNA replication. Valenciano studies a protein kinase that may be responsible for regulating proliferating cell nuclear antigen, a protein that coordinates the DNA replication and repair machinery through the process of phosphorylation. She has been able to successfully reproduce this process in vitro, meaning in a sample of biological molecules, but her next step is to determine whether this can be replicated in vivo, or in the actual parasite.

“The study of the relationship between these two protein regulators is important because it can be a significant contribution to the study of other neglected tropical diseases,” said Valenciano.

Ultimately Valenciano works to develop a deeper sense of the role this kinase plays in the parasite, which will also open up further possibilities for drug targeting.

Recently, both Ramsey and Valenciano completed a study with Mackey, which focused on the regulation of the PCNA protein within T. brucei. Specifically, their results show that this protein is tightly regulated--so much so that any alterations in its levels kills the parasite. The team’s next step is to understand how this happens in order to partner with chemists to synthesize small molecules, or drugs, that can target the disruption of this protein.

“Normally, PCNA belongs to a difficult class of targets for drug development, but Ana and Aaron’s hard work has revealed several new vulnerabilities that can make this target more amenable to exploitation,” said Mackey. “I’m very proud of their accomplishments.”

Written by Cassandra Hockman, communications coordinator for Fralin

Posted January 23, 2015