Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases
October 10th, 2020
Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.
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.
Overview
This protocol outlines a method for screening potential inhibitors of Glutathione S-transferases (GSTs), which are enzymes linked to chemotherapy resistance in cancer. The approach utilizes a spectrophotometric assay to evaluate the interaction between GSTs and various compounds.
Key Study Components
Area of Science
- Biochemistry
- Cancer Research
- Pharmacology
Background
- GSTs play a crucial role in detoxifying chemotherapeutic agents.
- Overexpression of GSTs is associated with resistance to cancer treatments.
- Inhibitors can potentially reverse this resistance.
- This study focuses on developing a reliable method to identify GST inhibitors.
Purpose of Study
- To provide a straightforward protocol for testing GST inhibitors.
- To determine the IC50 and Ki values for potential inhibitors.
- To analyze the mode of inhibition of GSTs.
Methods Used
- Preparation of GST enzyme solutions and substrate concentrations.
- Use of a 96-well plate for spectrophotometric measurements.
- Recording absorbance at 340 nm to assess enzymatic activity.
- Analysis of results using specialized software to calculate inhibition constants.
Main Results
- Successful determination of IC50 and Ki values for tested inhibitors.
- Identification of the mode of inhibition for curcumin and ethacrynic acid.
- Establishment of optimal assay conditions for GST activity.
- Application of the method to various GST isoforms.
Conclusions
- The protocol provides a reliable approach for screening GST inhibitors.
- Results can inform future cancer treatment strategies.
- Further studies can expand on the findings with different compounds.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables rapid, quantitative screening of GST inhibitors to address chemoresistance in oncology drug development. By providing IC50 and Ki determination alongside mode-of-inhibition analysis, it supports early-stage target validation and lead optimization. The method’s adaptability to recombinant human GST isoforms enhances its utility in preclinical de-risking strategies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of GST’s role in detoxification pathways and MAPK regulation, clarifying target biology in chemoresistance mechanisms.
- Operational Value: Provides standardized enzyme activity quantification (units/mL) and substrate kinetics (Km) for reproducible inhibitor screening across laboratories.
- Predictive Value: Competitive inhibition profiling (e.g., curcumin’s Ki) informs structure-activity relationships and prioritizes compounds with defined mechanisms.
Screening & Assay Development
- Assay Readiness: Establishes optimal GSH (25 mM) and enzyme (0.1 unit/mL) concentrations for consistent GST activity measurement in 96-well formats.
- Quantitative Output: Generates dose-response curves for IC50 determination and Lineweaver-Burk plots for Ki calculation, enabling potency ranking of inhibitor libraries.
- Scalability: Protocol details substrate titration (CDNB concentrations) and inhibitor dilution series, supporting adaptation to high-throughput screening platforms.
Translational & Preclinical Research
- Disease Relevance: Directly links GST inhibition to reversing resistance against electrophilic alkylating agents, a key mechanism in chemotherapy failure.
- Translational Continuity: Uses equine liver GST pool as a model for human isoforms, with noted applicability to recombinant GSTA1, GSTM1, GSTP1 for human-relevant validation.
- Mechanistic De-risking: Distinguishes competitive (curcumin) vs. other inhibition modes, predicting in vivo behavior and combination therapy potential.
Pipeline & Workflow Integration
Fits within early discovery to lead identification workflows, where enzymatic de-risking precedes cellular and in vivo validation of chemosensitizing agents.
- Discovery Biology: Supports hypothesis testing of GST’s dual role in detoxification and apoptosis regulation via measurable enzymatic outputs.
- Screening: Delivers reproducible, quantitative activity readouts (ΔA340/min) essential for assay robustness and inter-lab comparability.
- Analytics: Requires nonlinear regression for IC50 and secondary plots for Ki, providing statistical rigor for hit-to-lead progression.
- Translational Research: Connects enzyme-level inhibition to phenotypic outcomes in chemoresistance models only when supported by source (e.g., curcumin’s anticancer properties).
- Enterprise Reuse: Defined reagent concentrations (GSH, CDNB, enzyme) and plate layout enable standardization across GST isoform panels and compound libraries.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by requiring Km determination prior to inhibition testing, ensuring accurate Ki interpretation.
- Operational Value: Standardized 96-well plate workflow with defined timings (10-min kinetic reads) and blank controls enhances reproducibility.
- Strategic Value: Enables go/no-go decisions based on inhibition potency (e.g., curcumin’s IC50 ~31 µM) and mechanism, reducing investment in non-competitive or weak inhibitors.
- Portfolio Impact: Risk-adjusted prioritization of compounds demonstrating competitive inhibition with low Ki values, indicating sustained target engagement.
Implementation Considerations
- Requires expertise in enzyme kinetics and spectrophotometric data analysis using specialized software for Michaelis-Menten and regression modeling.
- Dependent on plate reader capable of 340 nm absorbance measurements and temperature control for kinetic stability.
- Necessitates cross-team agreement on unit definitions (e.g., 1 unit = µmol product/min/mL) and dilution protocols to ensure inter-assay consistency.
- Adaptation to recombinant GST isoforms requires validation of CDNB as a substrate (not applicable to GSTT1 per source).
- Practical limitation: Inhibitor solubility constraints (e.g., curcumin in water) may necessitate organic co-solvents, requiring matched blank controls.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:56
Preparation of the Enzymatic Solution
3:10
Michaelis-Menten Constant of CDNB with GST
4:42
Absorbance of Potential GST Inhibitors
6:08
Determination of the IC50
7:49
Determination of the Type of Inhibition and Ki
9:46
Results: Testing the Potential of Curcumin as a GST Inhibitor
13:56
Conclusion
