Abstract
Olfactory receptors (ORs) account for 49% of all G protein-coupled receptors (GPCRs), which are important targets for drug discovery, and hence ORs may also be potential drug targets. Various ORs are expressed in breast cancer cells; however, most of them are orphan receptors, and thus, their functions are unknown. Herein, we present an experimental strategy using a surface plasmon resonance (SPR) system and a cell-based assay that allowed the identification of orphan OR6M1 as a new anticancer target in the MCF-7 breast cancer cell line. After the construction of stable OR6M1-expressing cells, the SPR-based screening of 108 chemicals for ligand activity was performed against OR6M1-expressing whole cells (primary screening) or membrane fragments (secondary screening). As a result, anthraquinone (AQ) and rutin were discovered to be new OR6M1 ligands. Based on calcium imaging in OR6M1-expressing Hana3A cells, AQ and rutin were classified as an OR6M1 agonist and antagonist, respectively. Cell viability and live/dead assays showed that AQ induced the death of MCF-7 cells, which was inhibited by rutin. Therefore, OR6M1 may be considered an anticancer target, and AQ may be considered a chemotherapeutic agent. This combined method can be widely used to discover the ligands and functions of other orphan GPCRs.
Highlights
G protein-coupled receptors (GPCRs), which are expressed in the cell membrane, are the most widely used targets of drugs, including anticancer agents, with approximately 36% of FDA-approved drug targets [1]
Cell- and membrane-fragment-based screening approaches are indispensable in the study of orphan GPCRs, and this study effectively demonstrated the potential of these experimental methods
We presented an optimized strategy that shows the potential of orphan OR6M1 as a new drug target in the MCF-7 breast cancer cell line by integrating surface plasmon resonance (SPR)-based ligand screening and cell-based assays
Summary
G protein-coupled receptors (GPCRs), which are expressed in the cell membrane, are the most widely used targets of drugs, including anticancer agents, with approximately 36% of FDA-approved drug targets [1]. Since other orphan ORs expressed in breast cancer cells may have the potential to serve as pharmacological targets, ligands must be discovered for orphan ORs. Surface plasmon resonance (SPR) allows for the direct detection of protein–ligand interactions and is, a powerful tool for high-throughput screening (HTS) for novel ligands [10]. We present an experimental strategy that allowed the identification of human OR6M1, one of the orphan ORs expressed in MCF-7 cells, as a new anticancer target. The function of OR6M1 in MCF-7 cells was investigated using the discovered ligands This strategy can be used in the first phase of functional studies on orphan ORs and will be useful in confirming the potential of orphan GPCRs, including ORs, as drug targets
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