Characterization of Reversible Kinase Inhibitors using ... · Characterization of Reversible Kinase...

Characterization of Reversible Kinase Inhibitors using Microfluidic Mobility-Shift Assays

IntroductionCurrent drug discovery efforts typically focus on developing small molecule inhibitors of target enzymes involved in regulating specific cellular processes. Mechanism of action (MOA) studies are necessary to characterize and compare potential drug candidates. The ability to follow reaction rates in real time greatly facilitates the determination of inhibition mechanisms and the calculation of dissociation constants. With real time kinetics capability, Caliper’s LabChip® EZ Reader and LabChip 3000 provide ideal platforms for these studies. Individual wells within a microtiter plate can be sampled repeatedly during the course of an experiment, allowing for accurate determination of reaction velocities from the resulting reaction progress curves.

In addition, the mobility-shift format provides direct detection of both product and substrate to produce consistent high-quality data and minimize the potential for indirect effects of test compounds.

This application note describes MOA experiments conducted to characterize enzyme-inhibitor interactions for known inhibitors of the cyclic AMP-dependent protein kinase (PKA). Data obtained using 4-sipper and 12-sipper chips were used to demonstrate reversible inhibition, confirm rapid establishment of equilibrium between enzyme and inhibitor, determine inhibitor IC50 values at different substrate concentrations, discriminate between ATP- competitive, noncompetitive, or uncompetitive mechanisms of inhibition, and calculate Ki values.

Application Note 211

Table 1. PKA Mobility-Shift Assay conditions. Reactions were assembled as described for individual experiments in 384-well microtiter plates in a total volume of 71 µL.

Results and DiscussionIn order to make valid comparisons of relative activities between different classes of lead compounds, it is critical to characterize inhibitors with methods appropriate to the mechanism of inhibition. Figure 1 shows a flowchart outlining basic steps involved in lead characterization. After an initial IC50 determination, usually done at substrate concentrations equal to the Km value(s), studies are conducted to determine whether inhibitor binding is reversible, slowly reversible, or irreversible. In the case of irreversible inhibitors, evaluation of compound affinity should only be done after further characterizing the nature of binding between enzyme and inhibitor (right-hand side of Figure 1). For reversible inhibitors, reaction progress curves will show how rapidly the enzyme and inhibitor achieve equilibrium. Inhibitors that bind rapidly and produce linear progress curves can be evaluated using classical steady-state analyses. Inhibitors that bind the enzyme slowly and produce curvilinear progress curves should be evaluated with experiments designed to analyze time-dependent inhibition.

PKA is a well-characterized kinase with numerous commercially available inhibitors that have been analyzed for MOA. Previous work done with PKA, using the mobility-shift assay conditions described in Table 1, identified the apparent Michaelis-Menten constants and inhibitor IC50 values shown in Table 2. It is important to note that all constants determined when working with a kinase are apparent constants, because the enzyme catalyzes a two-substrate reaction. However, it is valid to treat a kinase reaction as a single-substrate reaction provided that only one substrate concentration is varied at a time, and the second substrate is kept constant at a concentration near its Km

app value.2

Application Note 211 - Page 7 - Application Note 211

Table 2. Kmapp and IC50 values used as initial estimates for

designing MOA experiments.

Figure 1. Biochemical analyses typically performed during the characterization of lead compounds for drug discovery. Adapted from Copeland, R.A. (2005)1


Reversible Inhibitor Binding Rapid dilution experiments were used to demonstrate reversible binding of H89 and Staurosporine to PKA. With this approach, inhibitor is allowed to bind enzyme at a concentration expected to inhibit 90% of activity, then diluted to a concentration expected to inhibit only 9% of activity. A reversible inhibitor dissociates quickly, allowing immediate recovery of approximately 91% of enzyme activity. An irreversible inhibitor prevents recovery of enzyme activity, and a slowly reversible inhibitor allows a gradual increase in activity.

For rapid dilution, 5 µL of 120X Enzyme (24 nM) was mixed with 1 µL of DMSO with and without inhibitor at 60X the IC50 value (H89: 1.8 µM, Staurosporine: 300 nM). After incubating 30 min at room temperature, the enzyme and inhibitor mixes were diluted 0.8 µL into 80 µL of 1X peptide (0.75 µM) and ATP (5 µM). The microplate was placed in the EZ Reader and wells were repeatedly sampled for 70 minutes. As shown in Figure 2, reactions containing H89 and Staurosporine proceeded at nearly 90% of the rate seen for control reactions, demonstrating reversible binding for both inhibitors.

Linear Progress Curves

Reaction progress curves were generated to demonstrate rapid establishment of equilibrium between PKA and its inhibitors. For this experiment, 1 µL 70X inhibitor in DMSO was spotted into each microtiter plate well. 17.5 µL of 4X ATP and 17.5 µL of 4X peptide were added and mixed with inhibitor. Reactions were initiated by the addition of 35 µL 2X enzyme, and progress curves were generated by repeated sampling of the reaction wells on the EZ Reader. Final concentrations were 32 µM ATP, 0.75 µM Peptide, 0.2 nM enzyme, 30 nM H89, 5 nM Staurosporine, and 0.6 nM PKI 6-22. The experiment was done with ATP at 8X the Km

app value, to ensure that the presence of excess ATP did not slow the establishment of equilibrium. As shown in Figure 3, the progress curves were linear through the first 20 minutes, giving no indication of time-dependent inhibition by any of these inhibitors. This result validates the use of standard steady-state analysis to determine inhibition mode with respect to the substrate ATP.

Figure 2. PKA activity was restored upon rapid dilution of concentrated enzyme-inhibitor mix.

Figure 3. Linear PKA reaction progress curves were observed in the presence of H89, Staurosporine, and PKI 6-22. GraphPad Prism 5 was used for linear regression of data from the first 20 minutes of the reaction. The slopes of the resulting lines and associated R2 values are shown in the table.


Steady-State Analysis of Inhibition Mode

To distinguish between ATP- competitive, uncompetitive, or noncompetitive mechanisms of inhibition, inhibitors were titrated at various concentrations of ATP. Figure 4 shows the plate layout for this experiment. Using one quadrant of a 384-well microplate, control reactions and an inhibitor titration are run at 6 different ATP concentrations, ranging from 8X Km to 0.25X Km. The inhibitor titration steps through a 9-point 1:3 dilution series starting at 81X the IC50 at ATP Km. Here, the reactions were run in kinetic mode, using a 12-sipper chip on the LabChip 3000, for the most accurate determination of initial reaction velocities. Alternatively, this experiment can be run as an endpoint assay by stopping reactions with the addition of EDTA.

Figure 4. Plate layout for steady-state analysis to determine inhibition mode with respect to ATP. For the experiments described in this note, the IC50 and ATP Km estimates shown in Table 2 were used to determine final concentrations of inhibitors and ATP. All reactions were set up by spotting 1 µL 70X inhibitor in DMSO, then adding 35 µL 2X enzyme, 17 µL 4X Peptide, and 17 µL 4X ATP. Final concen-trations of PKA and Peptide were 0.4 nM and 0.75 µM, respectively.

Figure 5 provides an example of kinetic data from reactions containing 32 µM ATP, and varying concentrations of H89 inhibitor. Using GraphPad Prism 5, the entire timecourse for each well was plotted as % conversion over time, and the first 5 data points were used for linear regression analysis. The chart shows the calculated slopes and the R2 value for each well.

To evaluate mode of inhibition, first the initial slopes were multiplied by the concentration of peptide substrate (0.75 pmoles/µL) and the volume of the reaction (70 µL) to obtain initial velocities in pmol product/min. Second, the formula % inhibition = 100*(1-((vi-background)/(v0-background))), where vi = velocity with inhibitor, v0 = average velocity of no inhibitor control, and background = velocity of no ATP control, was applied to the dataset. The % inhibition data was plotted vs. the log of ATP concentration and fitted in GraphPad Prism 5 with the formula for variable slope sigmoidal dose-response curves.

Figure 5. Example of kinetic data obtained from reactions at one substrate concen-tration. Linear regression analysis was used to determine initial reaction velocities.


Figure 6. Sigmoidal dose-response curves for H89 (A) and PKI 6-22 (B) fit to % inhibition data from reactions run with 6 different concentrations of ATP. Graphs C and D plot the IC50 values obtained from the curves shown in A and B vs. ATP concentration.

The dose response curves for H89 and PKI 6-22 are shown in Figures 6A and 6B. Plots of the calculated IC50 values vs. ATP concentration (Figures 6C and 6D) show patterns expected for competitive inhibition in the case of H89 and uncompetitive inhibition in the case of PKI 6-22.

Finally, initial velocity data was used for simultaneous non-linear regression analysis (SNLR) with models for competitive, uncompetitive and noncompetitive inhibition to determine best-fit values for Km, Vmax, and Ki or aKi. This analysis was done in GraphPad Prism 5, using built-in models for fitting the three equations shown in Table 3. Table 4 shows best-fit values for the variable parameters in each model, along with fit statistics. By comparing fit statistics, the model most closely describing the data was chosen. For H89, the model giving the R2 value closest to 1.0 and the lowest value for the sum of squares of residuals is that for competitive inhibition. For PKI 6-22, the best model is that for uncompetitive inhibition.

Figures 7A and 7B show the observed H89 and PKI 6-22 data points together with the curves generated from the appropriate equations with the best-fit parameters (Table 4, green columns). Graphs 7C and 7D map the distribution of residuals to illustrate how the actual data deviates from points predicted by the fitted equations. Note that data from higher inhibitor concentrations was not included in SLNR analyses, as data from reactions with low conversion levels was less reliable and reduced goodness of fit across all models.


Figure 7. Michaelis-Menten curves resulting from nonlinear regression analysis of initial velocity data with the equation describing competitive (A) and uncompetitive (B) inhibition. Graphs C and D show the distributions of residuals representing the differences between the observed initial velocities and the initial velocities calculated from the fitted equations.

Table 4. Results from SNLR analysis of H89 and PKI 6-22 data using the nonlinear fit function in GraphPad Prism 5. Parameter values are shown +/- standard error. The results from the model most closely fitting the data are highlighted in green.

Table 3. Equations used for SNLR Analysis of mechanism of inhibition in GraphPad Prism 5, where [S] = substrate concentration, v = initial velocity, and [I] = inhibitor concentration.

SummaryThis application note demonstrates the power of Caliper’s microfluidic assays for conducting mechanism of action studies. While the experiments shown were done with a kinase assay, the methods are ap-plicable to all enzymatic assays compatible with the mobility-shift platform. Real-time kinetics capability minimizes the number of reaction wells necessary for each experiment. Progress curves can be gener-ated rapidly to determine the reversibility of enzyme-inhibitor binding and the time required to establish equilibrium between enzyme and inhibitor. Steady-state analysis of mechanism of inhibition can also be done in kinetic mode, allowing reliable determination of initial reaction velocities for the most accurate estimation of best-fit model parameters. In addition, real-time kinetics capability enables a wide range of experiments necessary to explore more complex interactions between enzymes and inhibitors. Examples include use of tight-binding inhibitors to accurately determine enzyme concentration, characterization of tight-binding inhibition mode, and analysis of time-dependent inhibition.


References 1. Copeland, R.A. (2005) Evaluation of Enzyme Inhibitors in Drug Discovery, Wiley & Sons, Hoboken, New Jersey.2. Cornish-Bowden, A. (2004) Fundamentals of Enzyme Kinetics, Third Edition, Portland Press Ltd., London.

ITEM MANUFACTURER CATALOG NO.

LabChip EZ Reader I Caliper Life Sciences

ProfilerPro Chip, 4-sipper Caliper Life Sciences

LC3000 Drug Discovery System Caliper Life Sciences

Chip Module TC Caliper Life Sciences

Off-Chip Mobility-Shift Chip, Caliper Life Sciences 761037-0372R12-sipper, with coating reagent 3

PKA (catalytic subunit a, human, Upstate 14-440recombinant) Lot# 26698U, Specific Activity 11,430 U/mg

FL-LRRASLG-CONH2 Caliper Life Sciences 760095

HEPES, Free Acid ULTROL Calbiochem 391338

HEPES, Sodium Salt ULTROL Calbiochem 391333

Magnesium Chloride, hexahydrate Sigma M2670

Brij-35 Solution Sigma B4184

DTT EMD 3860

ATP, disodium salt Sigma A7699

EDTA, disodium salt, 0.5 M, pH 8.0 Ambion 9260G

DMSO JT Baker 9224-33

H89, Dihydrochloride Calbiochem 371963

PKI 6-22 Amide Calbiochem 539684

Staurosporine InSolution, Calbiochem 5693961mM in DMSO

18 MW water

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