SBIR-STTR Award

The objective of this proposal is to develop four-helix bundle analogs of G-Protein-Coupled Receptors (GPCRs), to accelerate structure-based drug development for these important proteins. GPCRs are integral membrane proteins of unknown structure that mediate cellular responses to diverse stimuli and are linked to many human diseases. A soluble four-helix bundle analog of a GPCR would facilitate structure determination and obviate requirements for lipids or detergents that currently hamper screening assays. In several GPCRs including the BETA2 adrenergic receptor (BETA2AR), determinants of ligand binding affinity and specificity arc concentrated in helices 3-6. Their orientation resembles that of soluble four-helix bundle proteins (4HB's). In Phase I BETA2AR-4HB will be constructed incorporating helices 3-6 and other key elements from BETA2AR. It will be optimized for stable expression in human cells, membrane localization, and specificity for agonists and antagonists. Disulfide bonds may be introduced to increase stability and a palmitation site added to ensure membrane anchoring. Phase II will involve engineering soluble variants of BETA2AR-4HB which couple to G-Proteins, and structure determination of membrane-bound and soluble forms. In Phase III, structural information will be used to identify lead compounds, and analogs of other GPCRs developed. PROPOSED COMMERCIAL APPLICATION: Generation of membrane-bound and soluble forms of the GPCR analog will greatly facilitate structure determination, and allow solution-based screens for agonists and antagonists. These advancements will accelerate drug discovery and structure based drug design efforts for the entire GPCR superfamily and will have a major impact on development of therapeutic agents for GPCR related diseases. GPCRs are targets for nearly 30% of drug discovery efforts worldwide, and over 60% of available drugs interact with a GPCR: estimated to be a $84 billion market in 1995

The objective of this proposal is to use four-helix bundle analogs of G- Protein-Coupled Receptors (GPCRs) to accelerate structure-based drug development for these important proteins. GPCRs are integral membrane proteins of unknown structure that mediate cellular responses to diverse stimuli, and are linked to many human diseases. Soluble analogs of GPCRs will facilitate structure determination and eliminate requirements for lipids or detergents that currently hamper screening assays. In several GPCRs including beta2 adrenergic receptor (beta2AR), determinants of ligand binding affinity and specificity are concentrated in helices 3-6. Their orientation resembles soluble four-helix bundles (4HB's). In Phase I a 4HB variant of beta2AR (beta2AR-4HB) was constructed, incorporating helices 3-6 from beta 2AR. It was expressed and shown to bind beta2AR effectors tightly and specifically. Phase II will involve engineering beta2AR-4HB for high level expression, and biochemical/biophysical characterization. Soluble variants of beta2AR-4HB will be constructed and adapted for high-throughput screening. The x-ray crystal structure of soluble and membrane forms will be determined. Analogs of beta3 adrenergic and dopamine receptors will be developed using the beta2AR-4HB paradigm. In Phase III, soluble variants of these and other GPCRs will be screened and structural information will be used to identify and refine lead compounds. PROPOSED COMMERCIAL APPLICATIONS: Determining the high-resolution structure of native GPCRs and analogs and generating soluble forms of these receptors will greatly facilitate screens for agonists and antagonists. These advancements will accelerate drug discovery and structure based drug design efforts for the entire GPCR superfamily and will have a major impact on development of therapeutic agents for GPCR-related diseases. In addition, high resolution structures of any GPCR, and the technology to generate soluble GPCR analogs will have a high value. GPCRs are targets for approximately 30% of drug discovery efforts worldwide. Over 60% of known drugs interact with GPCRs, creating a $84 billion market in 1995.