# KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers

## TL;DR

Researchers improved screening for drugs targeting potassium channels by creating better chimeric proteins that predict drug effectiveness more accurately.

## Contribution

The novel contribution is the design of KcsA-Kv1.x chimeras with complete ligand-binding sites for improved drug screening predictivity.

## Key findings

- T+F chimeras showed better peptide ligand-binding predictivity than T-only chimeras in phage ELISA assays.
- Electrophysiological data confirmed the filter region's role in establishing accurate relative affinity among toxins.
- The new chimeras provide a more reliable tool for drug development targeting Kv1.x channels.

## Abstract

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target–ligand interactions of these therapeutically relevant ion channels.