Rapid competitor data analysisusing GraphPad PrismTM

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Occasionally a compound dissociates too rapidly for the competition kinetics method to reliably quantify the dissociation rate.

A false fit results with unrealistically-high K3 and K4 values (see page 26). Effectively, the compound immediately equilibrates with free receptors, so no kinetic information is obtainable.

An alternative equation is available to analyze the data, that measures the equilibrium binding affinity.

Here the equation is applied in Prism,from GraphPad Software, Inc.https://www.graphpad.com/scientific-software/prism/

The equation is not one of the built-in equations in Prism but instead a custom equation.

A simple method is available to load equations, from a Prism file that already contains the equations.

Follow the steps outlined at the end of this guide, starting on page 27.

First, enter data into the data table.

Enter tracer binding data for each ligand concentration into separate columns.

Label the column header with the ligand concentration in nM. The analysis uses this number as a constant.

The rows are the different time points.

Enter specific binding data, i.e. data from which nonspecific binding has been subtracted.

Go to the graph of the data.

Now we are going to analyze the data.

The first step is to analyze the control data (0 compound, i.e. tracer alone without unlabeled compound).

Click “Analyze”

This brings up the “Analyze Data” dialogue

Prism defaults to analyzing all the data columns. We only want “0” so uncheck the other data sets.

Select “Nonlinear regression”

Click “OK”

This brings up the “Parameters: Nonlinear Regression” dialogue.

Now we select the equation.Click “Binding kinetics” checkbox.

Select “Association kinetics –One conc. of hot”

We need to enter some constants for the analysis to work.

To do this, go to the “Constrain” tab.

Enter the tracer concentration in nM.

Click “OK” to run the analysis.

K2 – dissociation rate constant of tracerFrom a separate experimentUnits of min-1 *

* Assuming the time unit in the data table is min. The time unit is the time unit of the data table.

Here is the fitted curve.

Now we need to retrieve K1.

Click “Nonlin fit” to bring up the results table

K1 is Kon

K1 = 2.46 × 107 M-1min-1

Now we are going to analyze the compound data.

Return to the graph

Click “Analyze”

Uncheck “0” in the data set list since we are analyzing only the compound data.

Select “Nonlinear regression”

Click “OK”

Open “User-defined” equations list.

This will only be available if you have loaded the custom equation already. See page 27 for instructions on how to load it from a file.

Select “[Pharmechanics] Kinetics of competitive binding, rapid competitor dissociation”

Now we need to enter some constants.

Go to the “Constrain” tab

We need to enter:

L – concentration of tracer in nM

K1 – association rate constant of tracerFrom the analysis of the control curveUnits of M-1min-1 *

K2 – dissociation rate constant of tracerFrom a separate experimentUnits of min-1 *

Note the compound concentration I in nM is entered as the column header of the data table.

Note Ki and Bmax are shared parameters. Prism fits data for all three curves simultaneously and fits a shared value.

* Assuming the time unit in the data table is min. The time unit is the time unit of the data table.

Here are the values for this particular experiment.

Click “OK” to run the analysis.

Here are the fitted curves.

Click “Nonlin fit” to bring up the results table

The “Global” values are the shared parameters.

The Ki value is in M

Ki = 4.0 × 10-8 M= 40 nM

“False fit” of rapid dissociation data when using the competition kinetics equation.

Very large error valuesUnrealistically high K3 and K4 values

0 30 60 90 1200

5

10

15

20

25

Time (min)

Spec

ific

trace

r bin

ding

033100300

Compound (nM)

Kinetics of competitive bindingBest-fit values K1 L K2 K3 I K4 BmaxStd. Error K3 K4 Bmax95% CI (profile likelihood) K3 K4 Bmax

33

= 24600000= 3.000= 0.05000~ 94395532404741= 33.00~ 316815543.16

~ 3.655e+016~ 12268798653.633

(Very wide)(Very wide)???

100

= 24600000= 3.000= 0.05000~ 94395532404741= 100.0~ 316815543.16

~ 3.655e+016~ 12268798653.633

(Very wide)(Very wide)???

300

= 24600000= 3.000= 0.05000~ 94395532404741= 300.0~ 316815543.16

~ 3.655e+016~ 12268798653.633

(Very wide)(Very wide)???

Global (shared)Ambiguous

~ 94395532404741

~ 316815543.16

~ 3.655e+016~ 12268798653.633

(Very wide)(Very wide)???

Good curve fits to the data

Loading equations into Prism from a file

GraphPad Prism contains an equation editor for the input of user-defined equations.

There is a sharing method that simplifies the loading of equations written by other users.

This avoids the need to write in the equation and all the fit settings.

This process is described in this presentation.

First, download the file containing the equations to your computer.

Then open it.

We are going to load the equation from the Results sheet.

Select the equation you want by clicking on the “Nonlin fit”

Results tab.

Click this icon

This process loads the equation into the “User-defined equations” list. It

only needs to be done once. After that, the new equation will be available

every time you open Prism.

Click “Details” for guidance on how to use the equation.

Experiment setup

Contact info

Data type

Analysis details

Click Close

Equation

Click OK then close file. Now the equation is available every

time you open Prism, in the “User-defined equations” list.