High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external...

Area of Science:

• Reproductive biology
• Pharmacology
• Ion channel research

Background:

• CatSper channels are exclusively expressed in sperm and crucial for sperm function, making them a prime target for male contraception.
• Developing effective male contraceptives is hindered by the lack of robust screening methods for CatSper inhibitors due to the channel's complexity.

Purpose of the Study:

• To develop and validate a high-throughput screening (HTS) assay for identifying novel CatSper channel blockers.
• To facilitate the discovery of new male, non-hormonal contraceptive agents.

Main Methods:

• A novel HTS assay was developed based on inducing CatSper-mediated monovalent cation influx by chelating divalent cations.
• The assay utilizes a membrane potential (Em) assay combined with fluorescent cell barcoding (FCB) for high-throughput flow cytometry.
• CatSper1 knockout (KO) sperm were used to confirm the CatSper-dependent nature of the observed depolarization.

Main Results:

• The developed assay successfully measures CatSper channel activity and drug-induced inhibition in mammalian sperm.
• Fluorescent cell barcoding significantly reduces acquisition times, enabling large-scale screening of live cells.
• The assay strategy was validated and applied to initiate high-throughput drug screening for CatSper blockers.

Conclusions:

• A validated, high-throughput screening method for CatSper blockers has been established.
• This assay provides a powerful tool for accelerating the discovery of novel male contraceptive drugs targeting CatSper.
• The developed methodology addresses the need for efficient screening of inhibitors for this complex ion channel.