Date of Award
Summer 2018
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
St. Maurice, Martin
Second Advisor
Hristova, Krassimira
Third Advisor
Yang, Pinfen
Abstract
Pyruvate carboxylase is a metabolically important enzyme that has been implicated in a wide range of diseases and infections such as cancer, diabetes and listeriosis. Despite the importance of pyruvate carboxylase in these diseases and infections, there are no selective and potent small molecule effectors available to study pyruvate carboxylase both in vitro and in vivo. Effective small molecule effectors of pyruvate carboxylase will expand the biochemical toolkit to facilitate the investigation of the structure, regulation and specific cellular role of pyruvate carboxylase. To discover small molecule effectors of pyruvate carboxylase, a novel fixed time assay was developed. The assay is based on the reaction of the diazonium salt, Fast Violet B, with the final product of the pyruvate carboxylase catalyzed reaction, oxaloacetate. This robust assay was validated and was used to screen numerous compounds in a high throughput format. In addition to high-throughput screening, a structure-based drug design project was implemented to specifically target the unique carboxyltransferase domain of pyruvate carboxylase, leading to the discovery of potent competitive inhibitors with respect to pyruvate. These compounds provide the foundation for chemical probes that target pyruvate carboxylase and have the potential to benefit a wide range of in vitro and in vivo studies that are investigating the role of pyruvate carboxylase in cancer, diabetes and listeriosis.