Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Wendy A Lea,Anton Simeonov,Paul C Driscoll

doi:10.1371/journal.pone.0036219

Wendy A Lea, Anton Simeonov + Show 1 more

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https://doi.org/10.1371/journal.pone.0036219

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Abstract

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization.

Highlights

A range of biophysical techniques are used to evaluate direct binding between a ligand and a target protein, and these can be based on calorimetry, surface immobilization, separation, or direct spectroscopic methods [1]
A method that overcomes some of these limitations is the fluorescence-based thermal shift assay, known as differential scanning fluorometry (DSF)
We examined the thermal stability changes of SjGST, hGST A1 and hGST M1 in the presence of three classes of inhibitors

Summary

Introduction

A range of biophysical techniques are used to evaluate direct binding between a ligand (most frequently, a small molecule) and a target protein, and these can be based on calorimetry, surface immobilization, separation, or direct spectroscopic methods [1]. A general method to evaluate compound-protein interaction is based on the ability of equilibrium binding ligand to perturb the protein stability upon application of a destabilizing factor, such as temperature, denaturing chemical, or proteolytic enzyme [1]. Many techniques, such as NMR, MS or calorimetry, can monitor ligand-induced protein perturbation, their utility is often limited by complexity and requirements for high protein consumption [1,2]. In DSF, an environmentally sensitive fluorescence dye whose quantum yield increases upon binding to hydrophobic protein regions is applied to monitor protein conformational stability upon thermal denaturation [3,4]. By coupling ligand binding to protein unfolding, protein Gibbs free energy of unfolding is increased, usually resulting in an increase in protein melting temperature, Tm, which in turn can be used as an indicator of a direct protein binder

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