An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR

Area of Science:

• Pharmacology
• Cell Biology
• Biochemistry

Background:

• Fluorescence imaging plate reader (FLIPR)-based assays are crucial for high-throughput screening (HTS).
• Continuous efforts are made to enhance the efficiency and accuracy of HTS methods.
• Identifying modulators of G-protein-coupled receptors (GPCRs) remains a key area in drug discovery.

Purpose of the Study:

• To develop and validate an optimized offline compound addition protocol for FLIPR-based HTS.
• To identify novel antagonists of the neurokinin-1 receptor (NK1R) using this improved screening platform.
• To assess the efficiency and throughput of the developed offline screening method.

Main Methods:

• Utilized a human astrocytoma cell line (U373 MG) expressing the NK1 receptor.
• Implemented an offline compound addition strategy using a 384-well plate format.
• Tested compounds in mixtures of 10 compounds/well, added offline before FLIPR assay.
• Stimulated NK1R with Substance P and measured intracellular calcium (Ca2+) flux.

Main Results:

• Substance P demonstrated a concentration-dependent Ca2+ increase with an EC50 of 0.30 ± 0.17 nM.
• Known NK1 antagonists L-733,060 and L-703,606 inhibited the Substance P-evoked response.
• The offline protocol enabled a throughput of approximately 200,000 compounds per 8-h day.
• Several novel structural classes of NK1 receptor antagonists were identified.

Conclusions:

• The offline compound addition protocol significantly enhances screening throughput for FLIPR assays.
• This method is effective for identifying modulators, specifically antagonists, of GPCRs like the NK1 receptor.
• The developed platform offers an efficient strategy for drug discovery and lead identification.