Date of Award

Summer 8-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

G proteins comprising of α subunit and βγ dimer are signaling proteins that play essential

roles in various pathological conditions. Direct modulation of these G proteins (specifically Gα subunit) using small chemical probes to elucidate their acute function is of great value. YM-254890 is a small molecule which is the first selective inhibitor of a class of G proteins, Gαq. However, despite its biological importance, this molecule is not available to researchers. In addition, the complex core structure of this cyclic depsipeptide has thwarted efforts to obtain a series of analogs by total synthesis. Moeller lab sought to overcome this obstacle by synthesizing simplified YM analogs that retain the ability to specifically inhibit Gαq. Thereby, this effort requires not only the synthesis of the YM analogs, but also the availability of both the purified G proteins and a rapid, cost effective method for screening newly synthesized analogs in real-time for their potential activity toward G proteins. This dissertation focuses on the 1) isolation and characterization of G proteins necessary to test the potency of simplified analogs and 2) development of a rapid screening method by utilizing the power of microelectrode arrays.

In Chapter 1 of this dissertation we discuss the potential utility of directly targeting G

proteins and why it is essential to develop G protein modulators. In Chapter 2, we provide

details on how three different G proteins (Gαq (wild type and mutant), Gαi1 and Gαo) were

isolated. While expression of recombinant proteins from insect cells is widely used, we applied the Titerless Infected-cells Preservation and Scale up method to express Gαq. A number of approaches were explored to optimize the biochemical assay that exhibits the activity of Gαq. Eventually a receptor-assisted nucleotide exchange assay was developed that could test the activity of purified Gαq. In Chapter 3, the activity of other G proteins was examined by a fluorescent nucleotide exchange assay. In addition, we introduce the first simplified analog of YM, WU-07047 and its potency towards Gαq and other G proteins was analyzed. Even though the receptor-assisted nucleotide exchange assay is a reliable way for testing the simplified analogs, it requires radio-labeled ligand and a number of accessory proteins. Hence, efforts were moved towards development of a rapid screening method utilizing the power of microelectrode arrays. The idea was to monitor binding interactions between immobilized small molecules and purified G proteins via electrochemical methods. Chapter 4 investigates an approach to modify the array surface via the use of PEG-polymer as a means to reduce non-specific binding. In addition to the reduction of non-specific binding, the ability to incorporate PEG onto the array

surface provides an opportunity to utilize PEG-polymers as a linker. These linkers move the immobilized molecule away from the array surface. In Chapter 5, we tested the compatibility of G proteins to the electroanalytical methods applied on microelectrode arrays. Moreover, we study a known binding interaction between a G protein and a short peptide on the arrays. Based on preliminary results, we can see specific binding interaction between them over non-specific background binding.

Language

English (en)

Chair and Committee

Kevin D Moeller

Committee Members

Kendall Blumer, Garland Marshall, Liviu Mirica, John-Stephen Taylor

Comments

Permanent URL: https://doi.org/10.7936/K7CV4G0K

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