HTS Technology for Discovery of Drugs Targeting GPCR Oligomers

描述(申请人提供)：GPCR家族调节体内神经递质和激素的生理作用，是FDA批准的50%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，GPCRs可以形成齐聚物，可以是同聚体，也可以是异构体。最近的研究表明，一些GPCR型杂寡体具有不同于单体或同源低聚体的配基特异性和功能反应。如果这是真的，这将为药物研究和发现开辟一个全新的领域，因为它将提供识别针对不识别单体的特定GPCR复合体的药物的可能性。这种低聚物导向的药物可能产生所需的药理作用，而作用于单体的药物缺乏组织或细胞选择性，这是目前制药业使用的药物发现方法所不可能的。我们建议开发一种新的技术，可用于针对GPCR寡聚体的药物的高通量筛选(HTS)。这种方法将是Patobios和DiscoveRx开发的技术的融合。Patobios已经证明，将核定位信号(NLS)插入GPCRs将受体从细胞膜引导到细胞质(O‘Dowd等人，2005年)。配体与GPCR的高亲和力结合导致GPCR保留在细胞膜上，提供独立于功能激活的配体结合读数。在初步研究中，我们显示了异源低聚物的内在化，其中一个GPCR被标记了NLS，而不同的GPCR没有标记。然而，如果配体与未标记NLS的受体结合，则复合体保留在细胞膜上，提供与GPCR杂低聚物结合的配体读数。DiscoveRx开发了一种敏感的酶片段互补(EFC)技术，可以使用发光读数以HTS格式检测GPCR的表面表达(Eglen和Singh，2003；Dunlop和Eglen，2004)。结合这些技术将提供一种筛选与异型低聚物唯一结合的药物的方法，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.