Project Summary A broad range of neurological indications, including schizophrenia, pain, Parkinson's and Alzheimer's diseases, and autism have been linked to G-protein coupled receptor proteins (GPCRs), making them attractive targets for therapy. Understanding GPCR dysfunction is essential for effective therapeutic development. However, deciphering the functions of GPCRs remains a daunting task in large part due to the inherent structural complexity of GPCRs, and the lack of tools/reagents for elucidating the GPCRs functions. Antibodies (Abs) can serve as highly specific analytical agents and have proven to be highly effective therapeutics. However, high- quality Abs against GPCRs are very difficult to make by traditional immunization-based methods. To address the unmet need of advanced tools for studying the functions of GPCRs, we propose to create a robust, integrated pipeline for the rapid discovery and characterization of selective, high-affinity nanobodies against defined GPCR structures of neurological importance using in vitro phase-display technology. We aim to overcome the two primary challenges to enable generation of renewable GPCR-binding reagents: (1) lack of functional GPCRs antigens availability, and (2) the inherent poor immunogenicity of GPCRs and high cost in producing GPCR-specific antibodies using conventional animal immunization method. Our approach is to leverage cell-free membrane protein synthesis method for facile preparation of large quantities of high-purity, biotinylated proteoliposomes (liposome-harboring GPCRs), which can be efficiently purified from reaction mixture by streptavidin-magnetic beads and used for parallel selection of nanobodies from phage-displayed synthetic nanobodies. By coupling in vitro GPCR production to in vitro antibody selection within the same laboratory, we anticipate construction of an integrated pipeline for routine generation of orthogonal GPCR- targeting nanobodies in a matter of weeks. In Phase I proof-of-concept demonstration, we will develop cell-free approach to synthesize cannabinoid receptors CB1 and CB2 and produce nanobodies to these two subtype receptors from phage-displayed nanobody libraries. The functionalities of resulting nanobodies will be characterized to determine binding affinity and specificity for the target GPCRs by biochemical and cellular methods. The receptors CB1 and CB2 are selected for demonstration due to their significant neurobiological functions. This proposed technology can be applied to other GPCRs in Phase II with an overall goal of 1) building libraries of GPCR proteoliposomes and GPCR-targeting nanobodies, 2) marketing the GPCR-targeting nanobodies as invaluable research reagents for studying GPCRs. Due to the unique properties of nanobodies, such as extremely robust, highly resistant to denaturation/thermal degradation, high aqueous solubility, and superior body distribution, tissue penetration, and the ability to cross blood-brain barrier, these nanobodies will provide invaluable new research tools for studying brain-derived GPCRs, and may have diagnostic and therapeutic utility. Given its modularity and scalability, this GPCR-nanobody pipeline has the potential to yield customized affinity reagents on a proteomic-wide scale, providing a high-quality and easy to use toolkit for GPCR functional analysis that will be broadly accessible to the biological and biomedical laboratories.

Project Narrative GPCRs have potential significance in healthy mental function and in the pathogenesis of several neurodegenerative diseases, and are the most common drug targets in neurological disease. Understanding GPCR dysfunction is essential for effective therapeutic development, but a large percentage of them remain poorly understood. The goal of this project is to create a scalable pipeline for generating GPCRs, and selecting their specific nanobodies for probing the functions of GPCRs in cells and tissues.