Poster abstracts

Poster number 102 submitted by Kevin McGarry Jr

Reengineering Human Butyrylcholinesterase for the Catalytic Degradation of Organophosphorus Compounds

Kevin G. McGarry, Jr. (OSBP), Yamin Fan (Chemical and Biomolecular Engineering), Anastasia C. Manesis (OSBP), Hannah S. Shafaat (Chemistry and Biochemistry), Danielle P. Perry (Ohio State University), David W. Wood (Chemical and Biomolecular Engineering)

Abstract:
From poisoning of agricultural workers, to acts of terrorism, to the battlefield, pesticide and chemical warfare nerve agent (CWNA) organophosphate compounds (OPs) are a continuing threat. It is estimated that as many as 200,000 people in the developing world die annually from acute OP poisoning, although most of these deaths are suicides. In the 1990s, in Japan two sarin attacks in the subway system resulted in the injury of approximately 1000 people. Further, CWNAs have been recently used by the Syrian regime on its own people during the ongoing civil war. It is widely understood that the acute toxic effects of OP poisoning involve cholinesterase inhibition; however, current therapeutic strategies for the treatment of acute OP poisoning are insufficient, and do not lend themselves to prophylactic applications.

Butyrylcholinesterase acts as a natural bioscavenger and can potentially be modified for use as a therapeutic. To reengineer BChE to perform as catalytic bioscavenger of OPs, we must first develop a platform for the synthesis, purification, and initial screening of mutant BChE enzymes. To do this we have worked to express human BChE in mammalian cell culture using HEK293, CHO, and, because BChE is naturally synthesized in the liver, we have begun development to utilize the hepatocyte cell line HUH-7. We have successfully demonstrated our ability to produce active BChE in multiple formats. Additionally, we have begun the initial characterization of a rationally designed hybrid metalloenzyme mutant BChE library whereby we have introduced a metal binding center into the active site of BChE. The reasoning for this approach is two-fold: 1.) Organophosphate hydrolase (OPH) is a bacterial metalloenzyme which hydrolyzes OPs and 2.) A mutant BChE (G117H) has shown OPH activity through the coordination of a water molecule for a nucleophilic attack on the OP. As metals can serve as a stronger nucleophile than water, our goal is to reengineer BChE as a metalloenzyme. Using our preliminary molecular modeling results and guided by the known structure of OPH, our goal is to introduce a metal binding site through rational site-directed mutation of residues near the catalytic triad. This novel BChE metalloenzyme may serve as a catalytic bioscavenger for the treatment of OP toxicity.

Keywords: BChE, organophosphate, chemical warfare