HTS Technology for Discovery of Drugs Targeting GPCR Oligomers

描述（由申请人提供）：GPCR家族介导体内神经递质和激素的生理作用，是50% FDA批准药物的治疗作用靶点。gpcr可以以多种形式存在。生化证据表明，gpcr可以形成低聚物，无论是同聚物还是异聚物。近年来的研究表明，一些GPCR异聚物与单体或同源低聚物相比，具有不同的配体特异性和功能反应。如果这是真的，这将开辟一个全新的药物研究和发现领域，因为它将提供识别针对不识别单体的特定GPCR复合物的药物的可能性。这种寡聚物导向的药物可能产生所需的药理作用，而这种作用于具有组织或细胞选择性的单体的药物是目前在制药工业中使用的药物发现方法所不可能达到的。我们建议开发一种新的技术，可用于针对GPCR低聚物的药物高通量筛选（HTS）。这种方法将融合由Patobios和DiscoveRx开发的技术。Patobios研究表明，将核定位信号（NLS）插入gpcr可使受体从细胞膜转移到细胞质中（O'Dowd et al, 2005）。配体与GPCR的高亲和力结合导致GPCR在细胞膜中保留，提供了独立于功能激活的配体结合读数。在初步研究中，我们发现了异聚物的内化，其中一个GPCR被标记为NLS，而另一个GPCR没有。然而，如果配体与未标记NLS的受体结合，则该复合物保留在细胞膜中，从而提供配体与GPCR异聚物结合的读数。DiscoveRx开发了一种灵敏的酶片段互补（EFC）技术，使用发光读数检测HTS格式的GPCR表面表达（Eglen和Singh, 2003； Dunlop和Eglen， 2004）。结合这些技术将提供一种检测方法来筛选与异聚物结合的药物，并导致新一代基于gpcr的药物的发现和开发。

DESCRIPTION (provided by applicant): The GPCR family mediates the physiological actions of neurotransmitters and hormones in the body and is a target for the therapeutic actions of 50% of FDA approved drugs. GPCRs can exist in multiple forms. Biochemical evidence suggests that GPCRs can form oligomers, either homomeric or heteromeric. Recent studies have indicated that some GPCR hetero-oligomers have different ligand specificity and functional responses than the monomers or homo-oligomers. If true, this would open up an entirely new field of drug research and discovery because it would offer the possibility of identifying drugs targeting specific GPCR complexes that do not recognize the monomers. Such oligomer-directed drugs might produce desired pharmacological actions lacking in drugs acting at monomers with tissue or cellular selectivity not possible with drug discovery approaches presently in use in the pharmaceutical industry. We propose to develop a novel technology that can be employed for high throughput screening (HTS) of drugs against GPCR oligomers. This approach will be a fusion of technologies developed by Patobios and DiscoveRx. Patobios has shown that insertion of a nuclear localization signal (NLS) into GPCRs directs the receptor away from the cell membrane to the cytosol (O'Dowd et al, 2005). High-affinity binding of ligands to the GPCR causes retention of the GPCR in the cell membrane providing a readout of ligand binding independent of functional activation. In preliminary studies we showed internalization of hetero-oligomers, in which one GPCR is tagged with an NLS and a different GPCR is not. However, if ligand binds to the receptor not tagged with the NLS, the complex is retained in the cell membrane providing a readout of ligand binding to GPCR hetero-oligomers. DiscoveRx has developed a sensitive enzyme fragment complementation (EFC) technology to detect surface expression of GPCR in a HTS format using a luminescent readout (Eglen and Singh, 2003; Dunlop and Eglen, 2004). Combining these technologies will provide an assay to screen for drugs uniquely binding to hetero-oligomers and lead to the discovery and development of a new generation of GPCR-based drugs.