Differential scanning fluorometry signatures as indicators of enzyme inhibitor mode of action: case study of glutathione S-transferase.

PloS One
Wendy A Lea, Anton Simeonov

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 obser...Continue Reading

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Citations

Jun 24, 2014·Ticks and Tick-borne Diseases·Rasha A GuneidyRagaa R Hamed
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Methods Mentioned

BETA
thermal shift
X-ray
PCR
differential scanning calorimetry
thermal shifts

Software Mentioned

GraphPad Prism
EXCEL

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