New Instrumentation and Methods for Analyzing Biological Compounds for Drug Discovery and Discovering Novel Disease Markers

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 limited to bulk solutions. To understand the liposome composition on a single vesicle/sensor level, a nano flow cytometer was built. This instrument uses a sheath flow cuvette combined with laser induced fluorescence detection for high sensitivity measurements of single vesicles. 200 nm and 400 nm DOPC vesicles doped with varying amounts of PE-CF lipid were measured. Single vesicle resolution was obtained through optimization of the sheath and capillary flow rates. These measurements revealed heterogenous lipid distribution within the same vesicle population. The single vesicle measurements using this instrument allow for more quantitative measurements when using fluorophores.