GPR75 is an orphan GPCR, meaning its endogenous ligand was initially unknown. Recent research has linked GPR75 to important physiological functions, including regulation of metabolism, appetite, and potentially neuroprotection. Its modulation could have significant therapeutic implications for a range of conditions, such as obesity, diabetes, and neurodegenerative diseases. Despite its potential, drug discovery efforts targeting GPR75 are still in relatively early stages. One major challenge is the lack of specific ligands and the limited understanding of the receptor’s structure and function. 

The GPR75-SRE-Luc reporter assay is a powerful tool in the drug discovery process. By using a luciferase reporter gene, this assay can measure the activation or inhibition of GPR75 in response to different compounds. This enables researchers to screen large libraries of compounds to identify potential drug candidates that modulate GPR75 activity. The use of medium-throughput screening allows for efficient evaluation of many compounds, accelerating the discovery process.

Mini Protocol

Here is the mini protocol for the GPR75-SRE-Luc reporter assay:

Cell Seeding:

1, Seed GPR75-SRE-Luc2P cells into a 384-well microplate using a MultiFlo dispenser.

2, Incubate the cells overnight to allow them to adhere and reach optimal confluence.

Compound Addition:

1, Add test compounds to the wells using an Echo655 acoustic liquid handler.

2, Incubate the cells with the compounds for 6 hours to allow interaction and potential modulation of GPR75 activity.

Luminescence Detection:

1, Add the Brightlight luciferase substrate to the wells using a Multidrop dispenser.

2, Measure the luminescence signal using a BMG FSX plate reader.

Assay Details

Number of Compounds: 1,000

Assay Format: Reporter assay (uses a luciferase reporter gene to measure GPCR activity)

Assay Type: Functional assay (measures cellular response to compound interaction)

Plate Type: 384-well microplate

Turnaround Time: 3 days

Automation: Available

Results

The luminescence results from the GPR75-SRE-Luc reporter assay are plotted in the figure above. The graph shows the relative luminescence units (RLU) for each compound tested, comparing the luminescence of the compound (cpd) treated wells to the DMSO control.

Red Dots (RLU cpd > DMSO): Compounds that increased luminescence compared to the DMSO control, indicating potential activation or agonism of GPR75.

Blue Dots (RLU cpd < DMSO): Compounds that decreased luminescence compared to the DMSO control, indicating potential inhibition or antagonism of GPR75.

Green Triangle (DMSO): Represents the baseline luminescence level with DMSO, the negative control.

Purple Triangle (Ref Compound): Represents a reference compound known to modulate GPR75, serving as a positive control.

Following the initial screening of potential GPR75 modulators using the HEK293T-SRE-Luc2P reporter assay, we identified several compounds that exhibited promising activity. These hits were subjected to a second round of validation to confirm their effects and ensure reproducibility.

Scatter Plot Analysis and Dose-Response Validation

Building on the results from our initial GPR75 medium-throughput screening (MTS), we conducted a deeper analysis focusing on the activation fold and chemical structure of the identified compounds. This scatter plot, as shown in the figure, highlights the relationship between the activation fold of each compound and its structural characteristics.

By analyzing the distribution of the compounds, we identified three key compounds—Cpd 1, Cpd 2, and Cpd 3—that exhibited different activation profiles and structural features. Following the identification of these three key compounds, we proceeded with dose-response studies to evaluate their potency and efficacy more comprehensively. The curves, illustrated in the accompanying graphs, confirmed the initial screening results, providing detailed insights into the activity of each compound across a range of concentrations.

This medium-throughput screening approach for the GPR75-SRE-Luc reporter assay effectively identifies potential modulators of GPR75. The use of automated systems, such as the MultiFlo and Echo655, ensures precision and efficiency, while the BMG FSX provides reliable luminescence readings. With a turnaround time of just 3 days, this protocol offers a rapid and robust method for early-phase drug discovery, highlighting ICE Bioscience's capability to support comprehensive compound screening programs.

The dose-response (DR) studies of the selected compounds provided valuable insights into their potential as GPR75 modulators. These findings underscore the importance of combining activation fold analysis with structural insights to refine compound selection. The successful validation of these compounds in DR studies reinforces their potential in the development of targeted therapies for conditions involving GPR75 modulation.

For more information on how our medium-throughput screening services can accelerate your drug discovery efforts, please contact ICE Bioscience.