SI - Discovery of the First Small-molecule Extracellular Inhibitor of KCa3.1

Background and Purpose:

The ion channel K_Ca3.1 plays a role in immune regulation, red blood cell function, and is linked to certain forms of cancer. Various animal toxins, such as maurotoxin, bind to the extracellular side of K_Ca3.1, providing a potential starting point for inhibitor development.

Experimental Approach:

To investigate new extracellular inhibitors, molecular dynamics simulations were performed using the experimental structures of K_Ca3.1 and maurotoxin. The simulations produced a validated binding mode, highlighting key residues involved in the interaction between the toxin and the channel. These findings laid the foundation for the structure-based identification of novel extracellular small-molecule inhibitors of K_Ca3.1. The Molport database, containing approximately 50 million compounds, was screened using protein-ligand docking, yielding a hit molecule that was experimentally confirmed using fluorescence-based and patch-clamp assays.

Key Results:

We report the discovery of a novel, small-molecule inhibitor, with an IC₅₀ of 58 µM, which was specifically designed to extracellular target plasma-membrane K_Ca3.1. In addition, the binding of peptidic toxin inhibitors like maurotoxin were modelled and validated via molecular dynamics simulations.

Conclusion and Implications:

The compound can serve as a starting point for the development of more selective inhibitors and probes.

What is already known?

·       K_Ca3.1 is a plasma-membrane and mitochondrial ion channel. Toxins selectively target the plasma-membrane channel and inhibit ion flow

·       Small-molecule inhibitors like senicapoc cross the membrane and target both channels

What does this study add?

·       Comprehensive investigation of computational design of novel extracellular inhibitors and in vitro validation

·       Small-molecule, drug-like tool compound that can be further developed into a selective K_Ca3.1 blocker

What is the clinical significance?

·       Start point for the development of a selective extracellular inhibitor that aids in the investigation of selective block of plasma-membrane K_Ca3.1