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

Background and Purpose: The ion channel KCa3.1 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 KCa3.1, 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 KCa3.1 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 KCa3.1. 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 KCa3.1. 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? · KCa3.1 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 KCa3.1 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 KCa3.1