A Solid Supported Membrane-Based Technology for Electrophysical Screening of B⁰AT1-Modulating Compounds

Area of Science:

• Biophysics
• Pharmacology
• Molecular Biology

Background:

• Classical high-throughput screening (HTS) assays for ionic currents often yield high false-positive rates due to indirect measurements.
• Electrophysiology-based assays, while direct, are typically complex and not suited for automated screening.
• There is a need for sensitive, direct, and automated methods for transporter-directed compound screening.

Purpose of the Study:

• To evaluate the suitability of solid supported membrane (SSM)-based electrophysiology for pharmacological compound identification and optimization.
• To assess the direct, automated screening capabilities of SSM electrophysiology for transporter proteins.
• To validate active compounds and identify false positives from primary screens using a direct assay.

Main Methods:

• Utilized cell-free SSM-based electrophysiological measurements.
• Focused on the electrogenic amino acid transporter B0AT1 (SLC6A19).
• Electrophysiological characterization, inhibition assays with a tool compound, and assessment of assay robustness and reproducibility were performed.

Main Results:

• Leucine-induced currents were specific to B0AT1, confirming target engagement.
• B0AT1 activity was successfully inhibited by an in-house tool compound.
• The SSM assay demonstrated robustness, reliability, and the ability to validate primary screening hits while identifying false positives.

Conclusions:

• SSM-based electrophysiology is a suitable direct method for rapid and automated identification of small molecules inhibiting B0AT1 activity.
• This technique offers an orthogonal approach to validate compounds identified through conventional HTS.
• SSM electrophysiology enhances the efficiency and accuracy of drug discovery for transporter targets.