Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases, GSTs, are metabolic enzymes that degrade intracellular electrophilic compounds and interact with MAP kinases involved in apoptosis pathway. Because of these two roles of expression of GST is correlated with drug resistance, a solution to counteract this issue is to use enzymatic inhibitors. With this protocol, we provide a way to test such molecules.

This method is aimed to everyone new to the field of GST inhibitors, who wish to utilize a quick and easy method for examining enzyme-compound's interaction. We also provide the steps to determine two characteristic of an inhibitor, the inhibitory concentration 50, IC50, and the constant of inhibition, Ki.As well we described the technique to define the mode of inhibition, an important feature that has different biological consequences. The first step is to determine the enzymatic activity of GST in solution per unit.

One unit per mil is the volume of enzyme needed to synthesize one micromolar of product per minute. Quantify the protein concentration of the enzyme solution. Choose the technique according to your convenience.

Dilute the protein solution to a final concentration of 0.02 milligrams per mil in water. Keep the enzymatic solution on ice. Prepare the reaction in a 96-well plate.

Add 20 microliters of water for the blank wells. Add 20 microliters of the enzymatic solution for the test wells. Add 20 microliters of GSH.

Add 150 microliters of PBS. Add 10 microliters of CDNB at 50 millimolar into each well. Mix the plate on a shaker for a few seconds.

With a spectrophotometric plate reader, record the absorbance at 340 nanometers every minute for 10 minutes. Analyze the results in a spreadsheet and with a specialized software. According to the results obtained, prepare a GST stock solution at 0.1 units per mil in water.

Prepare six different concentrations of CDNB 20 times in 95%ethanol in a final volume of 200 microliters. Into every well, add 20 microliters of GSH at 25 millimolar. To the test wells, add 20 microliters of the GST at 0.1 unit per mil.

To the blank wells, add 20 microliters of water. Into every well, add 150 microliters of PBS. To the test wells and the blank wells, add 10 microliters of the corresponding CDNB solution.

Every concentration of CDNB needs a specific blank. Mix the content on a shaker for a few seconds, record the absorbance at 340 nanometers every minute for 10 minutes. Analyze the results with a specialized software.

Determine the Km by drawing a Michaelis-Menten plot. According to the results, prepare a CDNB solution at 20 times the obtained Km in 95%ethanol. Dilute the inhibitor solution to the required concentration.

In a 96-well plate, add two microliters of the potential GST inhibitor. To the blank wells, add two microliters of the diluent. Add 20 microliters of GSH at 25 millimolar into every well and 168 microliters of PBS.

Add 10 microliters of CDNB 20 times Km found during the previous step. Mix the plate on a shaker for a few seconds. Record the absorbance at 340 nanometers every minute for 10 minutes.

Analyze the results with a specialized software. According to the results, the same blank can be used for each GST inhibitor or must be adjusted. Prepare nine concentrations of the tested GST inhibitor.

Prepare the essay solution consisting of 148 microliters of PBS and 20 microliters of GSH at 25 nanomolar for one reaction. Mix well of the solution. For the test wells, add two microliters of GST inhibitor, 20 microliters of GST, 0.1 unit per mil, and 168 microliters of the assay solution.

For the control wells, add two microliters of the diluent used for the GST inhibitor, 20 microliters of GST 0.1 unit per mil, and 168 microliters of the assay solution. For the blank wells, add two microliters of the diluent used for the GST inhibitor, 20 microliters of water and 168 microliters of the assay solution. Add 10 microliters of the CDNB solution for each well, including the blank.

Mix on the shaker for a few seconds. Record the absorbance at 340 nanometers every minute for 10 minutes. Calculate the IC50 with a specialized software.

Prepare four CDNB solutions at different concentrations as explained in the protocol. Prepare three GST inhibitor solutions at different concentrations, also as explained in the protocol. Prepare the enzymatic solutions containing 148 microliters of PBS and 20 microliters of GSH at 25 millimolar for one reaction.

For the control wells, add two microliters of the diluent used for the GST inhibitor. And for the test and blank wells, two microliters of the correct GST inhibitor solution. Mix for a few seconds.

Add 168 microliters of the enzymatic solution to each well. Add 10 microliters of the corresponding CDNB concentrations into each respective well. Mix for a few seconds, then record the absorbance at 340 nanometers every minute for 10 minutes.

Repeat the experiment using the same plating pattern in order to use the four different concentrations of CDNB along the three different concentrations of GST inhibitor. Analyze the results with a specialized software to determine the mode of inhibition, as well as the Ki.This protocol was applied to curcumin, a molecule with anticancer properties. This compound was selected by computational predictions of the binding affinity for several GST isoforms.

In parallel, the same experiments were conducted on ethacrynic acid, the most widely used GST inhibitor in the laboratory environment as a positive control. The inhibitory potency was tested on a pool of GSTs from equine liver and can be applied to any GST isoforms of choice. The IC 50 as well as the type of inhibition and the Ki were determined.

In order to provide the best assay conditions, first, Michaelis-Menten constant, Km of the substrate CDNB with a pool of GSTs was determined. Km is representative of the affinity between the substrate and the enzyme. The determination of this value is crucial in order to use a non-saturating concentration for the essay.

In fact, two high concentrations might disadvantage competitive inhibitors, whereas too little amount will not be detectable. Please see texts for more details on this. For this experiment, six different concentrations of CDNB were used with a fixed concentration of GSH and GST enzymes.

The Michaelis-Menten graph was obtained by plotting the substrate concentrations in millimolar on the X-axis and the velocity in millimolar per minutes on the Y-axis. Velocity of the reaction was calculated with equation one. Km is determined as half Vmax, a value obtained after saturation of the solution with the substrate in order to obtain a plateau of conjugate formation.

The Km of CDNB for the tested GST enzymatic solution was determined as 0.26 millimolar based on calculations with a specialized software. IC50 is defined as the concentration of substance needed to inhibit the enzyme activity by half. IC50 was estimated by using a nonlinear regression graph where the logarithmic concentration of the GST inhibitor was plotted on the X-axis and the percentage of GST activity on the Y-axis.

Nine different concentrations of inhibitor were used. The GST activity was determined with application of equation one. The results were normalized using the control with the no inhibitor.

Curcumin is poorly soluble in water. Thus higher concentrations were not possible to use, making it difficult to obtain maximum inhibition. Nevertheless, a specialized software was able to predict the IC50 of curriculum for this pool of GST at a value of 31.4 micromolar.

In parallel, the same experiment was conducted with ethacrynic acid as a positive control. As expected, this molecule was confirmed as a GST inhibitor with an IC50 of 6.7 micromolar. These results confirmed that curcumin is as well a GST inhibitor with an IC50 in the micromolar range, similar to ethacrynic acid.

To study more in detail curcumin as a GST inhibitor, the type of inhibition, as well as the constant of inhibition, Ki, were determined. First, the type of inhibition was assessed with the Michaelis-Menten graphs. By using different concentrations of the substrate CDNB whilst incrementing the concentrations of the GST inhibitor, Kms significantly increased in correlation with curcumin concentrations while Vmax remained unchanged despite the varying conditions.

This mode of inhibition is typical of a competitive mode of inhibition. For further details about the other types of inhibitions, please refer to the text. Ki was calculated accordingly based on the type of inhibition using the same dataset, providing a value of 23.3 micromolar of curcumin.

The explained methods are basic enzymatic essays, which require carefulness through certain steps in order to provide correct and reproducible results. Determination of the Michaelis-Menten constant Km, that delineates the affinity of the substrate to the enzyme is one of the crucial steps. In fact, when the substrate concentration is too high or too low, it makes it difficult to identify the correct mode of inhibition and Ki is not correctly calculated.

This procedure can be applied to human recombinant GSTs, as example GSTA1 and one of P1, are used in cell culture studies to assess the potential to be used in combination with electrophilic agents to reduce drug resistance.