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Orphan GPCRs G-protein‐coupled receptors (GPCRs) remain the most prolific class of drug targets for life-improving and life-saving medicines to-date. Of the 350 druggable non-sensory GPCRs, 120 remain “orphan”, as they lack identifiable native ligands. These orphan GPCRs (oGPCRs) represent vast opportunities for discovering new therapies for diseases that have been intractable by targeting the well-known GPCRs and other protein families. Although much progress has been made and published to uncover their tissue expression distributions, tools that can help understand the functions of these mysterious receptors remain scarce and limiting. Identifying chemical probes or surrogate ligands is an important first step. To do that, a robust high throughput screening (HTS) assay is critical for screening and validating surrogate ligands. The library interrogated can be small molecules, peptides, or antibodies.

MultiscreenTM β-Arrestin Sensor Various tools have been explored for establishing HTS assays for oGPCRs. Since G-protein coupling remains elusive for most oGPCRs, it is advantageous to employ assays independent of G-protein cell-signaling pathways. Therefore, β-arrestin recruitment and receptor internalization become attractive assay options. However, intracellular-tagging of GPCRs at their c-termini is often required using older technologies to detect the β- arrestin pathways. This can lead to skewed assays results. To overcome these barriers, Multispan has newly developed MultiScreenTM β-Arrestin Sensor technology, making β-Arrestin HTS possible for native GPCRs without tagging in a homogeneous assay format. 

oGPCR HTS Assay With tools such as MacMam viral particles and β-Arrestin-expressing parental stable cell line clones, available at Multispan, oGPCRs cell-signaling can now be tested transiently for proof-of-concept. This can be quickly followed by stable cell line generation and assay development for de-orphanization HTS campaigns in a high throughput manner. To help jumpstart your research, Multispan offers 75 full-length orphan GPCR cDNAs that have been cloned and sequence-validated in the patented MultiScreenTM pMEX2 vector (Table 1). pMEX2 is specifically designed for robust surface expression of GPCRs in mammalian cells, such as HEK293T, CHOK1, NIH3T3, HELA and many other cell lines. By way of an example, a pilot panel of 5 oGPCRs has been tested after transient transfection in HEK293T cell line clones stably expressing β-arrestin 1 and β-arrestin 2 (Table 2 and Figure1). All 5 oGPCRs show β-Arrestin2 signaling whereas 4 show coupling through β-Arrestin1. We can now take these transiently validated oGPCRs to further develop HTS-ready stable cell lines and conduct de-orphanization HTS using MultiScreenTM β- Arrestin Sensor with confidence. Please stay tuned! Inquire: info@multispaninc.com

Table 1: Orphan GPCRs Cloned in Patented MultiScreenTM pMEX2 GPCR-Expression Vector

Table 2: Transiently Transfected oGPCRs Assayed in MultiScreenTM β-Arrestin stable cell line Clones

Figure 1: Transiently Transfected GPR18 Assayed in MultiScreenTM β-Arrestin Parental Stable Cell Line

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