How to Use Inhibitors

Read on to learn tips and tricks on how to successfully use inhibitors including how to select the right inhibitor for your experiment and how to successfully plan experiments with inhibitors.

• How to Select the Right Inhibitor
  
  • Tips for Selecting Inhibitors
  • Examples for Selecting Inhibitors
• How to Plan Experiments With Inhibitors
  • Tips and Tricks to Successfully Use Inhibitors in Assays
  • How Much Inhibitor Should You Use?
• Related Inhibitor Products

How to Select the Right Inhibitor

Before starting an experiment with inhibitors, you must ask some critical questions. 

Tips for Selecting Inhibitors

Tips to select the right inhibitor include:

• Determine what molecule(s) is (are) important for disruption of function.
  
• Determine if a broad-spectrum inhibitor or specific inhibitor is suitable for the particular experiment.
• Look at other enzymes potentially inhibited and the concentrations at which they are inhibited.
  • For example, H-89 is a good inhibitor for protein kinase A (IC₅₀ = 48 nM). However, at much higher concentrations it will also inhibit protein kinase C (IC₅₀ =31.7 μM); myosin light o   chain kinase (K_i = 28.3 μM); Ca²⁺/calmodulin-dependent protein kinase II (K_i = 29.7 μM); and casein kinase I (K_i = 38.3 μM).
• A lower IC₅₀ means a more potent inhibitor. Consider the IC₅₀ of the inhibitor. Is toxicity a concern?
• Determine if a cell-based inhibitor is needed or not. Cell permeability is the primary consideration for cell-based inhibitor studies.
• Determine if the inhibitor should be reversible or not. Cell permeability and reversibility can limit the selection of inhibitors.
• Determine if the mechanism of action is of concern— competitive, non-competitive, etc. It may be more important to first understand the mechanism of the selected inhibitor.
• How pure is the inhibitor? Purity of 98% by HPLC is much better than 98% purity by TLC.
• Check to see if the reconstitution solvent is compatible with the assay.
  • Most cells will tolerate up to 1% dimethylsulfoxide (DMSO). Solvents such as methanol, acetonitrile, dimethylformamide (DMF) are toxic to cells and will affect their viability.
• Check the vial label to see if it should be protected from light or moisture. In some cases, exposure to light or moisture may damage the molecule.
• Use our Bioactive Small Molecules search tool to locate inhibitors or contact your local technical service scientist for recommendations.

Examples for Selecting Inhibitors

Here are a few examples relating to the selection of inhibitors in various common applications. These are only a few examples, and for your specific experimental models, please contact our technical service team or refer to suitable published literature.

Selection of a Protein Kinase C Inhibitor

• To reversibly inhibit a wide range of phosphorylation events affecting serine/threonine residues in live cells, a broad range, cell-permeable inhibitor, Staurosporine (Product No. 569397) can be used. Staurosporine inhibits most of the known serine/threonine protein kinases.
• To inhibit all isozymes of protein kinase C in live cells, then a PKC-specific, cell-permeable inhibitor, such as Bisindolylmaleimide (Product No. 203290) should be selected. It has a very low IC₅₀ value (low nM range) and will inhibit all PKC isozymes.
• In the next step, to inhibit only the Ca²⁺- dependent isoforms of PKC in live cells, then Gö6976 (Product No. 365250) can be used. It has very low IC₅₀ values (2 to 10 nM) for PKC α, β, and γ isozymes and it does not affect non-Ca²⁺-dependent PKC isozymes.

Criteria for Selecting a Protease Inhibitor

When processing cells or tissues, it is critical to assume that active proteases are present in the medium or are being secreted. Hence, it is important to include protease inhibitors even in the early steps of sample preparation. For best results, add protease inhibitors to the medium just prior to harvesting. The use of inhibitors in buffers stored over a period of time is not recommended.

Different cells and tissue types exhibit different protease profiles.

• Serine proteases are widely distributed in all cells
• Bacterial cells contain higher levels of serine and metalloproteases
• Animal tissue extracts are rich in serine-, cysteine-, and metalloproteases
• Plant extracts contain higher quantities of serine and cysteine proteases

If you are not sure of the type of proteases present in the sample, it is best to use a readily available inhibitor cocktail or customize your own cocktails.

Table 1. Protease inhibitors classified by their target class.

Protease Type	Inhibitor Examples
Aspartyl proteases	Acetyl-pepstatin; Pepstatin
Cysteine proteases	Antipain; Calpastatin; Calpeptin; Cathepsin inhibitors; Chymostatin; Cystatin; Leupeptin
Metalloproteases	1,10-phenanthroline; Bestatin; EDTA/EGTA; Phosphoramidon
Serine proteases	AEBSF; Antichymotrypsin; Antipain; Antithrombin; Antitrypsin; Aprotinin; Benzamidine; Bovine pancreatic trypsin inhibitor (BPTI); DFP; Ecotin; Leupeptin; PMSF; TLCK; TPCK

Determining If a Caspase Inhibitor is Reversible or Irreversible

• The C-terminal group determines the reversibility or the irreversibility of any caspase inhibitor.
• In general, caspase inhibitors with an aldehyde (CHO) group are reversible.
• The CMK, FMK, and FAOM groups are more reactive and form covalent bonds with the enzyme, creating an irreversible linkage.
  • FMK is slightly less reactive than CMK and therefore is considered more specific for the enzyme site being inhibited.

Advantages of Using FMK- Based Caspase Inhibitors and How They Differ From CHO-Based Inhibitors

FMK-Based Caspase Inhibitors:

The FMK-based caspase inhibitors are cell-permeable because the carboxyl group of aspartic or glutamic acid is esterified. This makes them more hydrophobic. These inhibitors covalently modify the thiol group of the enzyme, making them irreversible inhibitors. Generally, at the amine end of the inhibitor, there is a Z, biotin, or Ac group. These groups also increase the hydrophobicity of the molecule, which makes them more cell permeable. Compared to the inhibitors with an Ac or a biotin group, those inhibitors with a Z group are even more cell permeable. Inhibitors with a biotin group can serve as a detection tool and are useful in tagging the enzyme‘s inhibitor binding site.

CHO-Based Caspase Inhibitors:

The CHO-based inhibitors are different from FMK-based inhibitors and are reversible because the thiol group of the enzyme forms a reversible adduct to the carbonyl group of the aldehyde. As a general rule, CHO-based inhibitors are hydrated and hence are slow-binding. The extent of their reversibility depends on the pH, metal ion concentration, and other conditions. When the aldehyde group is attached to the aspartic acid (D-CHO), the product exists as a pseudo acid aldehyde in equilibrium. This makes the inhibitor somewhat cell permeable.

How to Plan Experiments With Inhibitors

To quantify enzyme efficiency, biochemists perform a series of experiments to monitor the initial reaction rate and determine the V_max of the reaction.  To reduce the rate of reaction and obtain reliable measurements, use reversible or irreversible inhibitors. Below are a few recommendations to follow when using inhibitors in a biological or biochemical assay.

Tips and Tricks to Successfully Use Inhibitors in Assays

• Decide on your sample preparation method (whole cell, lysate, tissue homogenate, etc.), inhibitor permeability, and potential sensitivity of your sample to toxicity before you plan your experiments.
• The precise time of inhibitor addition and the duration of treatment are important in the interpretation of data.
  • An inhibitor can be added at time zero along with substrate and enzyme (Figure 1A)
  • It can also be added to an ongoing enzyme-substrate reaction (Figure 1B)
  • An enzyme can also be preincubated with an inhibitor for a designated period of time prior to the addition of substrate (Figure 1C)
  • Theoretically, if the enzyme is totally inhibited, no product should be generated. Each of these reaction protocols will give different results and have to be interpreted differently. For any comparison, data should be derived under identical conditions.
    • For example, if an inhibitor is added to an ongoing reaction (Figure 1B), the reaction rate will decline. However, some substrate has already been converted to end product. These results cannot be compared to an experiment where an inhibitor is added at time zero. In the latter case, much less product will be formed than if the inhibitor is added to an ongoing reaction.

Figure 1. Effect of timing of inhibitor addition on enzyme activity. A. No preincubation with inhibitor. B. Addition of inhibitor to an ongoing reaction. C. Preincubation with inhibitor.

• To compensate for nonspecific effects of the inhibitor, it is essential to run a control reaction adding only the solvent used for inhibitor reconstitution.
  • For example, if an inhibitor is dissolved in DMSO, then, with each set of experiments, a DMSO control should be run simultaneously.
• When multiple samples are run and data are used for comparison, it is important to terminate the reaction at the same time points.
  • For example, if 10 samples are run simultaneously and the incubation period is 10 minutes for each sample, it is important to time the reaction in a way that each sample is exposed to an inhibitor for exactly 10 minutes. Additional incubation of just one minute may add a 10% error to the data. Exact timing is particularly critical with shorter incubations and when studying the time course of any reaction.
• It is possible that, throughout an experiment, particularly when the incubation period is long, the pH of a weak reaction buffer may change. Hence, it is best to select a buffer with optimum strength to overcome minor changes in pH.
• An enzyme and reaction product can undergo degradation over time. Hence, extrapolating data may not be appropriate.
  • For example, the outcome of reaction after 60 minutes cannot be deduced from the results of a 5- or 10-minute incubation. In a time-course experiment, it is best to run parallel experiments for each time point or pipette out small volumes of the reaction mixture at multiple time points.
• If experiments are being conducted on tissue homogenates or cell lysates, then the release of nonspecific proteases can destroy part of the enzyme. This will add experimental error because the amount of active enzyme in the initial phase of reaction (or at early time points) will be greater when compared to later time points. Hence, it is best to add an appropriate protease inhibitor cocktail to the reaction mixture to avoid any nonspecific protease activity.
  • Our ready-to-use Protease Inhibitor Cocktail III (Product No. 539134) is highly recommended for use with mammalian cells and tissues.
• To normalize the results of an enzyme reaction to a specific amount of protein, it is best to set aside a small volume of sample for protein analysis. Then, calculate the reaction as μg or mg substrate converted or product formed per mg protein per unit time.
• The method used to terminate a reaction should be given some consideration, depending on how the reaction product is analyzed. In most cases, perchloric acid (PCA) or trichloroacetic acid can be used. However, in some cases, these acids will interfere with further sample analysis and it may be necessary to neutralize these samples.
  • However, neutralizing with an alkali will generate more salt, which must be removed by an appropriate method. When conducting experiments with substrate labeled with a radioisotope, the reaction can be slowed down by adding an excessive amount of “cold” non-radioactive substrate, which will reduce the incorporation of radioactivity into the end product. It is best to process samples as soon as possible or freeze them (-20 or -70°C) for later analysis.

Find answers to common questions about preparing inhibitors for experiments in our FAQs article.

How Much Inhibitor Should You Use?

The amount of inhibitor required depends on various factors such as:

• Accessibility
  
• Cell permeability
• Duration of incubation
• Type of cells used

It is best to survey the literature to determine the initial concentration. If published K_i or IC₅₀ values are known, then use 5 to 10 times higher than these values to completely inhibit enzyme activity. If K_i or IC₅₀ values are unknown, then try a wide range of inhibitor concentrations and use Michaelis-Menten kinetics to determine the K_i value. When 20-fold or higher concentrations of inhibitors are used, it is not unusual to see either no inhibitory effect or a reverse effect. Always run an appropriate control to compensate for nonspecific effects of solvent used to solubilize the inhibitor. 

Learn more about important terms and calculations to know before you start your next experiment.

For Research Use Only. Not For Use In Diagnostic Procedures.