シグナル伝達タンパク質の機能を模倣：人工シグナル伝達療法に向けて

The overall goal of this experiment is to spectroscopically follow the unnatural communication between two proteins induced by a synthetic chemical transducer. The will demonstrate the possibility of developing synthetic analogs of signalling proteins that will be able to carry out signal transduction pathways not found in nature. The ability to follow unnatural protein to protein communication by common spectrophotometer could help develop and improve the function of artificial chemical transducers and bring us closer to achieving artificial signal transduction therapy.

The chemical transducer is composed of a platelet derived growth factor, or PDGF aptamer. A bivalent ethacrynic amide GST inhibitor, a fluorophore and a quencher. The synthesis of the chemical transducer will not be demonstrated in this video.

But is described in the text protocol. The operating principles of the chemical transducer are illustrated here. The chemical transducer inhibits the enzymatic activity of GST, when the two EA groups bind the enzymes active sites.

Addition of PDGF leads to the formation of the PDGF chemical transducer complex. Which disrupts the transducer-GST interaction, therefore restoring GST enzymatic activity. The following addition of an unmodified PDGF aptamer releases the chemical transducer and allows it to re-inhibit the enzyme.

Prior to starting the experiments for examining control of GST activity by PDGF, set up an experimental procedure in the plate reader for kinetic measurement. Create a new experiment as a standard protocol. Select Procedure to open the Procedure Settings window.

In the pop-up list on the upper side of the window, choose the 384 Plate Type according to the plate manufacturer. Select Read on the left menu. Choose Absorbance for the Detection Method.

And Endpoint for the Read Type. In the Wavelength window, type 340 nanometers. Select Full Plate, and choose the wells to be measured.

Then click OK to close the Read window. On the left menu, choose Start Kinetic. Set the Run Time as 10 minutes.

Select the Minimum Intervals option. Then click OK to close the Kinetic window. Drag the Read Line into the Kinetic Measurement.

Select the Validate button, followed by the OK button. Save the experiment. Select the Play button.

A dialog box will appear, but do not select the OK button until ready to start the kinetic measurement. Once the plate reader has been set up, the next step is to prepare the samples for measuring GST activity in the presence of the chemical transducer and PDGF. In order to test four concentrations of PDGF in triplicate, prepare four samples where each contains 3.25 microliters of the chemical transducer, and 3.25 microliters of GST M-1.

Add to each sample, zero, 1.2, 2.4 or 4.9 microliters of PDGF respectively. Then add the appropriate volume of the assay buffer to each sample to bring the total volume to 130 microliters. Incubate the samples at room temperature for 10 minutes.

After 10 minutes, transfer 40 microliters of each sample to the wells of a 384 transparent well plate. Transfer each of the four samples to either the odd or the even wells in the same row only. To allow the use of a multi-pipetter for substrate addition.

Next, use a 12-channel multi-pipetter to quickly add to each sample 10 microliters of each substrate previously prepared in a 96 well plate. Mix quickly, but gently, to avoid bubbles. Immediately insert the plate into the plate reader, and start the kinetic measurement by selecting the OK button in the dialog box.

To examine GST activation inhibition cycles mediated by the chemical transducer, prepare five samples, each containing 84.5 microliters of assay buffer. 3.25 microliters of chemical transducer, and 3.25 microliters of GST M1-1. Incubate at room temperature for three minutes.

Add 3.65 microliters of assay buffer to sample one, and 3.65 microliters of PDGF to samples two, three, four and five. Incubate at room temperature for three minutes. Add 3.12 microliters of assay buffer to samples one and two, and 3.12 microliters of PDGF aptamer to samples three, four and five.

Incubate at room temperature for three minutes. Add 24.4 microliters of assay buffer to samples one, two and three. And 24.4 microliters of PDGF to samples four and five.

Incubate at room temperature for three minutes. Add 7.8 microliters of assay buffer to samples one to four, and 7.8 microliters of PDGF aptamer to sample five. Incubate at room temperature for five minutes.

Transfer the samples to a 384 transparent well plate. To perform the experiment in triplicate, and quickly add the substrate to the samples. Insert the plate into the reader, and start the kinetic measurement.

To begin this assay, set up an experimental procedure in the plate reader for kinetic measurement as described in the protocol text. Prepare two samples in two wells of a 384 transparent well plate. For each well, mix one microliter of GST M1-1 and one microliter of the chemical transducer in 38 microliters of assay buffer.

Leave an empty well between the two wells. Using a 12-channel multi-pipetter, quickly add 10 microliters of substrate to each well. And mix gently and quickly to avoid bubbles.

Immediately insert the plate into the reader and start the kinetic measurement. When the plate reader opens up after 3.5 minutes, quickly add 1.125 microliters of PDGF to one of the wells, mix gently, and allow the plate to re-enter the reader for the remaining kinetic measurements. Prior to preparing the samples for this experiment, set up an experimental procedure in the plate reader for kinetic measurement as described in the protocol text.

A nitrite/nitrate colorimetric kit will be used for measuring nitric oxide production. In a 96 well plate, dispense 50 microliters of assay buffer per well into the first row. 70 microliters of Griess One Reagent per well into the second row.

And 70 microliters of Griess Two Reagent per well into the third row. Prepare four samples, each containing 4.8 microliters of the chemical transducer. To sample one, add 155.2 microliters of assay buffer.

To sample two, add 3.2 microliters of GST M1-1, and 152 microliters of assay buffer. To sample three, add 9.6 microliters of PDGF, and 145.6 microliters of assay buffer. And to sample four, add 3.2 microliters of GST M1-1, 9.6 microliters of PDGF, and 142.4 microliters of assay buffer.

Incubate the samples at room temperature for 10 minutes. Dispense 50 microliters of sample per well in a 384 transparent well plate to perform the experiment in triplicate. Dispense samples only to the odd or even wells in the same line to allow the use of a multi-pipetter for substrate addition.

Add 0.54 microliters of JS-K prodrug to each sample. Quickly add to each sample 10 microliters of glutathione solution and mix gently and quickly to avoid bubbles. Insert the plate into the reader, and start the kinetic measurement.

Immediately after the kinetic measurement, transfer 50 microliters of each sample to each well of the first row of the 96 well plate with assay buffer. Then transfer 50 microliters from each well of the second row of the plate into the wells of the first row of the plate. And 50 microliters per well from the third row of the plate into the wells of the first row of the plate.

Incubate while protecting from light for 10 minutes at room temperature. Before measuring the absorbance at 550 nanometers. Measuring GST activity without the chemical transducer, and with the chemical transducer showed that the chemical transducer inhibits GST activity.

GST is reactivated by the addition of increasing concentrations of PDGF to the GST-chemical transducer complex. Sequential additions of PDGF, and an unmodified PDGF aptamer to the GST-chemical transducer complex revealed that the communication between PDGF and GST was reversible. The addition of PDGF to the GST-transducer complex resulted in a rapid increase in GST activity, 3.5 minutes after adding substrates.

While the addition of a PDGF aptamer to a mixture of GST, PDGF and the chemical transducer resulted in a decrease in GST activity. These results indicate that the system responds in real-time to changes in its environment. Subsequently, the ability of this system to control prodrug activation was investigated using JS-K, an anti-cancer prodrug that is activated by GST to release toxic nitric oxide.

Measurements of nitric oxide amounts released upon the addition of JS-K to the chemical transducer and different combinations of GST and PDGF confirmed that only the presence of both GST and PDGF activates JS-K. This all shows how a synthetic agent can enable a growth factor to trigger the catalytic activity of Glutathione S-Transferase, which is not its natural enzyme partner. Future biomimetics of this class may therefore be used to alter the response of cells to environmental signals or provide them with new properties.