(131f) Tether Supported Biomembrane-Microsphere Assemblies for Drug Discovery
AIChE Annual Meeting
Monday, November 9, 2009 - 4:45pm to 5:03pm
Our primary objective is to develop a highly miniaturized, solid-phase platform for assaying the function and inhibition of integral membrane proteins (IMPs). The vast majority of clinical drug targets are membrane proteins and yet these molecules have not been widely built into array formats suitable for high throughput screening. Native (homologous) cellular expression levels are often very low (<500 IMPs/cell), and despite major advances, recombinant production remains a challenging, trial-and-error task due to complexities in expression, folding and isolation.
To functionalize materials with active membrane proteins, the challenge is to build stabilized, supported biomembranes in which the substrate to biomembrane spacing can be controlled to accommodate larger membrane protein systems such as the ABC transporter protein. These structures have been employed as a route to functionally immobilize the yeast drug efflux pump PDR5 (as a GFP fusion), an important anti-fungal drug target. These assemblies are being used to build in vitro models of multidrug resistance for high-throughput screening and drug discovery.
The core of the methodology is to create a biomimetic, surface-tethered artificial cytoskeleton where polymer bioconjugates anchor the biomembrane stucture. The silica microsphere (5 micron) surface was functionalized with DiNHS-PEG in order to make a tethering bridge designed to yield a spacing of 6-10 nm from the silica. Purified PDR5 membranes were tethered onto these PEG-functionalized silica particles using fusion and also detergent-based reconstitution. Confocal fluorescence microscopy was utilized to analyze the silica surface at different stages of surface modification and examine the passivation of the substrate to the formation of untethered lipid bilayers. Fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching (FRAP). Fluorescence Activated Cell Sorting (FACS) was used as a quality control step to purify the PDR5-GFP proteolipobead complexes. Transport studies employing in situ confocal microscopy in parallel with flow cytometry were used to form prototypical inhibition assays.