Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology.

Krisztina Pesti,Adam V Toth,Katalin Zboray,Mátyás C Földi,Arpad Mike,Peter Lukacs

doi:10.3389/fphar.2021.738460

Abstract

We have developed an automated patch-clamp protocol that allows high information content screening of sodium channel inhibitor compounds. We have observed that individual compounds had their specific signature patterns of inhibition, which were manifested irrespective of the concentration. Our aim in this study was to quantify these properties. Primary biophysical data, such as onset rate, the shift of the half inactivation voltage, or the delay of recovery from inactivation, are concentration-dependent. We wanted to derive compound-specific properties, therefore, we had to neutralize the effect of concentration. This study describes how this is done, and shows how compound-specific properties reflect the mechanism of action, including binding dynamics, cooperativity, and interaction with the membrane phase. We illustrate the method using four well-known sodium channel inhibitor compounds, riluzole, lidocaine, benzocaine, and bupivacaine. Compound-specific biophysical properties may also serve as a basis for deriving parameters for kinetic modeling of drug action. We discuss how knowledge about the mechanism of action may help to predict the frequency-dependence of individual compounds, as well as their potential persistent current component selectivity. The analysis method described in this study, together with the experimental protocol described in the accompanying paper, allows screening for inhibitor compounds with specific kinetic properties, or with specific mechanisms of inhibition.

Highlights

In silico prediction of drug effects can save a tremendous amount of time and resources, and can accelerate drug discovery
In the accompanying paper (Lukacs et al, 2021) we describe how different degrees of inhibition observed in the 17-pulse protocol can be interpreted as revealing the state-dependent onset (SDO; how the inhibition depended on the length of depolarizations), the recovery from inactivation (RFI; how the inhibition depended on the length of hyperpolarizations), and steady-state inactivation (SSI; how the inhibition depended on the membrane potential)
In this study we focus on micro-dynamics

Summary

Introduction

In silico prediction of drug effects can save a tremendous amount of time and resources, and can accelerate drug discovery. To predict the therapeutic action of sodium channel inhibitors, an elementary knowledge about the mechanism of action is essential These drugs show state-dependent accessibility and affinity to binding sites and can have radically different binding/unbinding kinetics. The beststudied example for this is the torsadogenic effect of certain compounds in the human heart, where in silico modeling of multi-target effects is a generally accepted directive (Sager et al, 2014) This initially has been done by determining the IC50 value of a specific drug to all relevant ion channel targets, from which the inhibited fraction of specific ion channels can be determined at specific drug concentrations. It is possible that if we increase the concentration of compound “B", the onset will be just as fast as that of compound “A.” It is possible that if we increase the concentration of compound “A,” it will delay recovery just as effectively as compound “B.” We aimed to find compoundspecific (and concentration-independent) properties of inhibition, and it turned out that each compound did have such properties, resting and inactivated state affinities (KR and KI), but more importantly the kinetics of approaching KR upon hyperpolarization, and approaching KI upon depolarization were such compound-specific properties

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Discussion

Conclusion