Date of Award

Spring 5-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Glucose transport is a fundamentally important process for maintenance and regulation of cellular metabolism in all kingdoms of life. Despite their high importance, detailed examination of glucose transport proteins in humans and parasites through biochemical, biophysical and structural properties was greatly hampered by the inability to express, purify and reconstitute sufficient amounts of active transporters. This dissertation describes strategies that led to the first successful expression, purification, stabilization and functional reconstitution of active insulin-responsive GLUT4 transport protein. Furthermore, the work described herein establishes a requirement of anionic and conical lipids for full activity of the mammalian glucose transporters GLUT3 and GLUT4, thereby extending the field of known membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers. Because of its crucial role in parasite survival, the malarial glucose transporter PfHT has been extensively validated as drug target for different parasitic life stages in vitro and in animal models. The emergence of parasites with resistance to even the most potent existing anti-malarial drugs has made paramount the development of novel drugs that target essential pathways for parasite survival. We identified PfHT as molecular target of the antimalarial activity of the clinically used HIV inhibitor lopinavir which had been shown previously to decrease parasite viability in vitro, in vivo, and in patients. In order to find novel PfHT inhibitors with increased potency and selectivity over human orthologs, a high-throughput assay was developed that uses fluorescence as direct readout of PfHT mediated glucose transport inhibition. Validation of this approach was demonstrated by our success in identifying several verified hits in a screen of the MMV malaria box compound library. Importantly, we identified a potent PfHT inhibitor with >10 fold higher selectivity for PfHT over its human orthologs. These findings have high potential for direct application in large-scale screens and new drug development. Taken together, this work provides a novel framework for ongoing efforts to directly target glucose transporters in the treatment of human disease.

Language

English (en)

Chair and Committee

Paul W. Hruz

Committee Members

Colin G. Nichols, Katherine Henzler-Wildman, Weikai Li, Paul H. Schlesinger,

Comments

Permanent URL: https://doi.org/10.7936/K7C827KP

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