An Intracellular Allosteric Modulator Binding Pocket in SK2 Ion Channels Is Shared by Multiple Chemotypes

Abstract

Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.

A Pilot Study of All-Computational Drug Design Protocol–From Structure Prediction to Interaction Analysis

“…We first constructed the human SK2/calmodulin model (PSK2) using SWISS-MODEL server and docked the ligands Riluzole and CyPPA analog 1 (PDB ligand ID: 658, see section 2.1), onto the predicted binding site. The predicted site is consistent with the reported results (Cho et al, 2018). Then the residues V481 and A477 in the binding pocket were virtually mutated and the mutation effects were assessed via binding energies (MM-GBSA G Bind ) calculation.…”

“…It was also reported that ligands of two different chemical classes, Riluzole and its analog NS309, and CyPPA and its analogs, all bind to the same binding site where the interface of SK2 and calmodulin is. In addition, two key residues in the binding site were mutated to investigate how different mutations affect the potency of two ligands (Cho et al, 2018). As a proof of concept, we chose SK2 as the target to examine the protocol we proposed.…”

“…As a proof of concept, we chose SK2 as the target to examine the protocol we proposed. Hence, in this study, we basically repeated the entire procedure of the previous study of Cho et al (2018) with consecutive in silico approaches and compared our results with those of the bench experiments. We first constructed the human SK2/calmodulin model (PSK2) using SWISS-MODEL server and docked the ligands Riluzole and CyPPA analog 1 (PDB ligand ID: 658, see section 2.1), onto the predicted binding site.…”

Concomitant SK current activation and sodium current inhibition cause J wave syndrome

“…It does not activate Ca 2+ and other voltage-dependent K + currents; however, it causes an inhibition of BK and Na + currents (12). CyPPA binds to and allosterically modulates via a binding pocket situated at the intracellular interface of SK and calmodulin, which increases the SK/calmodulin's affinity for Ca 2+ (19). Due to overlapping activation ranges between SK3 (EC 50 = 5.6 ± 1.6 μmol/l) and SK2 (EC 50 = 14 ± 4 μmol/l) and the possibility of forming heteromeric complexes (20,21), whether CyPPA at the current concentration can activate heteromeric channels remains unclear.…”

Ataxic Symptoms in Huntington’s Disease Transgenic Mouse Model Are Alleviated by Chlorzoxazone

“…injections with CHZ converted bursting PCs to tonic spike generation state in aging SCA2-58Q animals (Egorova et al, 2016). Another chemical compound riluzole attracts and allosterically activates the SK2 channel molecules (Cho et al, 2018). A randomized, double-blind, placebo-controlled trial on a mixed population of ataxia patients with different forms of hereditary cerebellar ataxia demonstrated that the exposure to 100 mg/day riluzole alleviated the SARA scores in patients and did not reveal any life-threatening toxicity (Romano et al, 2015).…”