A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors.

Area of Science:

• Pharmacology
• Biotechnology
• Molecular Biology

Background:

• G-protein-coupled receptors (GPCRs) are crucial drug targets.
• High-throughput screening for GPCR agonists is vital for pharmaceutical development.
• Functional assessment of GPCRs coupled to G alpha(s) or G alpha(i/o) proteins is challenging.

Purpose of the Study:

• To report the use of a novel, high-density, well-less screening platform, microARCS, for identifying novel GPCR agonists.
• To demonstrate a method for functionally evaluating GPCRs coupled to G alpha(i/o) proteins using Ca(2+) release.
• To screen a large compound library for novel agonists of the human dopamine D(4.4) receptor.

Main Methods:

• Utilized microarrayed compound screening (microARCS) to test 8640 compounds per plate.
• Engineered HEK-293 cells to stably coexpress the human dopamine D(4.4) receptor with a chimeric G(alpha qo5) protein, enabling Ca(2+) release detection.
• Preloaded cells with fluo-4, cast them into agarose gel, and imaged fluorescence response using a ViewLux CCD system.

Main Results:

• Successfully screened a library of 260,000 compounds using the microARCS platform.
• Identified several novel agonists for the human dopamine D(4.4) receptor.
• Observed time- and concentration-dependent increases in fluorescence response to dopamine and other agonists.

Conclusions:

• The microARCS platform provides enhanced efficiency and throughput for GPCR agonist screening.
• This technology enables functional evaluation of GPCRs not amenable to standard high-throughput assays.
• microARCS serves as a valuable alternative platform for identifying novel GPCR agonists, accelerating drug discovery efforts.