The overall goal of this procedure is to screen small molecules for botulinum neurotoxin, a light chain inhibition. This is accomplished by first preparing compound dilution series with high accuracy and precision. The second step is to transfer compound dilutions into a black 96 well plate.
Next diluted light chain enzyme is added to the plate and the enzyme is incubated with the compound for five minutes at room temperature. The final step is the addition of the light chain fret substrate to initiate the reaction, which is monitored with a fluorescence plate reader. Ultimately, secondary screens that utilize a non fluorescent peptide substrate should be employed to confirm compound activity and determine more rigorous kinetic constants such as the inhibitor dissociation constant or ki.
The main advantage of this assay over existing methods such as HPLC based or cell-based methods, is that it is simple to perform, easily automated, and can be used to compare the relative potency of a number of compounds. Individuals may struggle when new to this method when preparing a compound dilution series and properly mixing the plate once all the components are added. Begin this procedure by preparing the buffers and reagents as described in the text protocol.
Configure the plate reader to shake the plate at medium speed for 10 seconds, and then to monitor the fluorescence at an excitation wavelength of 490 nanometers and an emission wavelength of 523 nanometers every five minutes in kinetic mode for one hour 30 minutes at room temperature. After determining the compound concentration range label tubes with the appropriate compound name and concentration, Eloqua, 100%dimethyl sulf oxide or DMSO to all of the tubes to prepare them for compound cereal dilution. Next, prepare serial dilution of the compounds for the standard one to three dilution series.
Add two microliters of the undiluted compound to four microliters of DMSO in the first tube of the dilution series, and mix well with a new pipette tip. Remove two microliters of the first dilution and add to four microliters of DMSO in the second tube mix. Well repeat for the remaining dilutions.
Make sure to mix the compound dilutions well to ensure that the compound concentrations are accurate. Cover the compound tubes and set them aside At room temperature, obtain a flat bottom, opaque black 96 well plate. Using the recommended plate setup that can be found in the text protocol.
Manually dispense one microliter of the respective compound dilution directly to the bottom of each corresponding well for wells one through nine, dispense one microliter of the highest concentration of compound to be tested to well.11. To serve as the compound intrinsic fluorescence control. This control is to determine if the compounds themselves are fluorescent and or affect substrate hydrolysis.
Manually dispense one microliter of a known potent inhibitor to well, 12 as a positive control and one microliter of 100%DMSO to well. 10 as the negative control with an automated pipetter dispense. 79 microliters of assay buffer to the side of wells, one through 10 and 12 and 89 microliters to the side of well, 11.
Make sure not to touch the tip to the bottom of the well where compound is present to avoid cross-contamination of the wells. Vortex, the diluted light chain enzyme, and use an automated pipetter to add 10 microliters of the enzyme to the side of each well, except for well 11. Gently tap the plate to ensure that the total volume of added enzyme goes to the bottom of the well gently mix, cover, and incubate the plate for five minutes at room temperature.
This incubation provides a pree equilibrium step for the compounds to bind to the light chain. Prior to substrate addition, gently vortex the botulinum neurotoxin a light chain substrate. Then using an automated pipette, add 10 microliters of substrate to the side of each.
Well be sure to add the substrate to the side of the well opposite of where the enzyme was added earlier. Gently tap the plate to mix the reagents immediately. Place the plate in a fluorescence microtiter plate reader and begin the previously configured program.
After the program is finished, calculate the enzyme velocities by graphing the relative fluorescence unit versus time and calculating the slope of the line during the linear period of the reaction. Prepare for automated operation for high throughput screening as described in the text protocol, Demonstrating a portion of the high throughput screening protocol will be de young a postdoc in my laboratory. He will show how compounds are transferred into plates for high throughput screening using laboratory automation equipment After dispensing diluted botulinum neurotoxin, a light chain enzyme into the assay plate.
Assign areas on the liquid handler platform for the compound plates, assay plates, and containers with cleaning solutions. Program the liquid handler to place the compound plates and the assay plates in the designated positions and remove the plate lids prior to stamping. Calibrate the stamping program software and ensure that the stamping pins are submerged but not scratching the bottom of the assay plate.
Stamp 50 nanoliters of the compound directly into the assay plate containing eight microliters of botulinum neurotoxin. A light chain. Clean the pins after each plate by dipping them in DMSO and then drying on blotting paper and repeating this process with isopropanol and methanol.
Finally, dry the pins over the fan after the compounds have been stamped in the assay plates with enzyme. Separately, stack the stock compound plates and the assay plates cover the top assay plate in the stack to prevent evaporation and set aside. Make sure to incubate the light chain with compounds for at least five minutes at room temperature.
Longer incubation times can be used when stamping a large number of plates. Finally, dispense the substrate into the assay plates and measure the fluorescence as described in the text protocol. Whether performing the fret based botulinum neurotoxin a light chain assay in a low throughput or high throughput manner, an increase in fluorescence should be observed over time when the botulinum neurotoxin, a light chain is incubated with the substrate.
If serial dilutions of a compound are tested, then a series of lines with varying slopes is often obtained.Here. The final concentration of a hydroxy ate based compound is listed in micromolar units. Well, 10 in the plate layout described here serves as a negative control where vehicle only is added.
The rate of this reaction is defined as 100%Enzyme activity and rates in the presence of compound can be normalized to this rate to obtain the relative rate or percent inhibition. When the assay is performed with serial dilutions of a compound, a graph of the rate of the reaction versus the inhibitor concentration can be used to determine the half maximal inhibitory concentration or IC 50. The dilution range should be chosen so that the plot appears sigmoid shaped for optimal curve fitting.
The IC 50 value is a relative measure of potency best described as an apparent KI value as it depends upon the concentration of enzyme present in the assay. This value can then be used to compare and rank the potency of several compounds if tested. In parallel, proper mixing of the plate after adding each component is crucial to obtain a smooth linear increase in fluorescence over time, which is necessary to accurately determine the initial velocities.
This plot shows a representative experiment where there was insufficient mixing, and the rate of product formation is not linear over time. Confounding data analysis Once mastered this technique, can be done in less than two hours for up to eight compounds. After this experiment, other assays can be performed with non fluorescent peptide substrates to confirm that compounds inhibit botulinum neurotoxin, light chain, and somatic activity.
After watching this video, you should have a good understanding of how to determine the relative potency of botulinum neurotoxin light chain inhibitors. The protocol was accomplished over three steps. First, the preparation of a compound dilution series.
Second, the addition of recombinant light chain enzyme, and third, the addition of a fluorescent substrate and subsequent measurement on a fluorescent plate reader.
