New Instrumentation and Methods for Analyzing Biological Compounds for Drug Discovery and Discovering Novel Disease Markers
type: CreativeWork
description: Ligand-receptor interactions mitigate intracellular responses, with abnormal signaling corresponding to different disease states. G-protein coupled receptors currently compose 50% of drug targets, signifying their importance in the pharmaceutical industry. Therefore, monitoring the binding events of ligands to receptors within the cell membrane is highly important for drug development. One key limitation of accurately monitoring ligand binding to a transmembrane protein is the reconstitution of the protein. Many platforms lack conformational freedom for reconstituted membrane proteins, affecting ligand binding measurements. This work developed a new liposome shell microparticle platform to tether lipid vesicles, which provide conformational freedom for the protein. These particles can be used in a pull-down assay for identification of ligand-receptor binding pairs. CHO-K1 cell membranes containing 5-HT1A receptors were tethered to sulfonate modified particles. NAN-190, a fluorescent 5-HT1A antagonist, binding to 5-HT1A receptors was monitored using flow cytometry. Liposome shell microparticles may also be used in packed bed membrane-based liquid chromatography. To retain the particles a frit is needed within the column. In this work a new thermal polymerization method for fabrication of on-column frits was developed. This method has advantages including a short polymerization time, ease of frit placement within the capillary, minimal and inexpensive equipment and retention of the polyimide capillary coating. Thermal frits were synthesized within a packed bed column and allowed for separation of phenolic acids and aliphatic amines. Frit stability and reproducible retention times demonstrate the utility of this new method for frit synthesis. Lipid vesicle heterogeneity may also affect ligand binding. Lipid membranes can be doped with fluorescent lipids and/or fluorophores can be encapsulated within sensors. However, liposome composition measurements are currently limi…
abstract: Ligand-receptor interactions mitigate intracellular responses, with abnormal signaling corresponding to different disease states. G-protein coupled receptors currently compose 50% of drug targets, signifying their importance in the pharmaceutical industry. Therefore, monitoring the binding events of ligands to receptors within the cell membrane is highly important for drug development. One key limitation of accurately monitoring ligand binding to a transmembrane protein is the reconstitution of the protein. Many platforms lack conformational freedom for reconstituted membrane proteins, affecting ligand binding measurements. This work developed a new liposome shell microparticle platform to tether lipid vesicles, which provide conformational freedom for the protein. These particles can be used in a pull-down assay for identification of ligand-receptor binding pairs. CHO-K1 cell membranes containing 5-HT1A receptors were tethered to sulfonate modified particles. NAN-190, a fluorescent 5-HT1A antagonist, binding to 5-HT1A receptors was monitored using flow cytometry. Liposome shell microparticles may also be used in packed bed membrane-based liquid chromatography. To retain the particles a frit is needed within the column. In this work a new thermal polymerization method for fabrication of on-column frits was developed. This method has advantages including a short polymerization time, ease of frit placement within the capillary, minimal and inexpensive equipment and retention of the polyimide capillary coating. Thermal frits were synthesized within a packed bed column and allowed for separation of phenolic acids and aliphatic amines. Frit stability and reproducible retention times demonstrate the utility of this new method for frit synthesis. Lipid vesicle heterogeneity may also affect ligand binding. Lipid membranes can be doped with fluorescent lipids and/or fluorophores can be encapsulated within sensors. However, liposome composition measurements are currently limi…
author: Sandy, Kendall Elizabeth
language: en
keywords: analytical chemistry, ligand discovery, particle, pull-down assay
date published: 2018
date modified: 2026-07-24T00:56:22Z
provider: University of Arizona
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: The University of Arizona.

PropertyValue
property ID: Handle
value: http://hdl.handle.net/10150/628482